Process for the preparation of tricyclic PI3K inhibitor compounds and methods for using the same for the treatment of cancer

ABSTRACT

The present disclosure provides for methods for preparing tricyclic PI3K inhibitor compounds in high yield and purity in aqueous solvent systems.

RELATED APPLICATIONS

The present disclosure is a continuation of U.S. Non-Provisional patentapplication Ser. No. 15/780,328, filed 31 May 2018, and claims priorityto International Patent Application Serial No. PCT/US2016/067174, filed16 Dec. 2016, and published as International Patent ApplicationPublication No. WO 2017/106647, which claims the benefit and priority toU.S. Provisional Application Ser. No. 62/291,248, filed 4 Feb. 2016; andU.S. Provisional Application Ser. No. 62/288,832, filed 29 Jan. 2016;and U.S. Provisional Application Ser. No. 62/268,149, filed 16 Dec.2015, each of which is incorporated herein by reference in theirentirety.

FIELD OF THE DISCLOSURE

The disclosure relates generally to methods for preparing compoundswhich inhibit PI3 kinase activity. The disclosure also relates tomethods of using the compounds for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, or associated pathologicalconditions. The disclosure also relates to methods of treating cancercharacterized by the overexpression of PI3 kinase.

BACKGROUND

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of the common general knowledge in the field.Phosphatidylinositol is one of a number of phospholipids found in cellmembranes which play an important role in intracellular signaltransduction. Cell signaling via 3′-phosphorylated phosphoinositides hasbeen implicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al. (1999) J. Biol Chem, 274:8347-8350). The enzymeresponsible for generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PBK),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al.(1992) Trends Cell Biol 2:358-60).

Phosphoinositide 3-kinases (PBK) are lipid kinases that phosphorylatelipids at the 3-hydroxyl residue of an inositol ring (Whitman et al.(1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s)generated by PB-kinases act as second messengers recruiting kinases withlipid binding domains (including plekstrin homology (PH) regions), suchas AKT and phosphomositide-dependent kinase-1 (PDK1). Binding of AKT tomembrane PIP3s causes the translocation of AKT to the plasma membrane,bringing AKT into contact with PDK1, which is responsible for activatingAKT. The tumor-suppressor phosphatase, PTEN, dephosphorylates PIP3 andtherefore acts as a negative regulator of AKT activation. The PB-kinasesAKT and PDK1 are important in the regulation of many cellular processesincluding cell cycle regulation, proliferation, survival, apoptosis andmotility and are significant components of the molecular mechanisms ofdiseases such as cancer, diabetes and immune inflammation (Vivanco etal. (2002) Nature Rev. Cancer 2:489; Phillips et al. (1998) Cancer83:41).

The main PI3-kinase isoform in cancer is the Class I PI3-kinase, p110α(alpha) (see, e.g., U.S. Pat. Nos. 5,824,492; 5,846,824; 6,274,327).Other isoforms are implicated in cardiovascular and immune-inflammatorydisease (Workman P (2004) Biochem Soc Trans 32:393-396; Patel et al.(2004) Proceedings of the American Association of Cancer Research(Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA;Ahmadi K and Waterfield M D (2004) Encyclopedia of Biological Chemistry(Lennarz W J, Lane M D eds) Elsevier/Academic Press). The PI3kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such modulating or inhibitory agents would be expectedto inhibit proliferation, reverse the repression of apoptosis andsurmount resistance to cytotoxic agents in cancer cells (Folkes et al.(2008) J. Med. Chem. 51:5522-5532; Yaguchi et al. (2006) Jour, of theNat. Cancer Inst. 98(8):545-556).

Malignant gliomas are the most common primary brain tumors in adults. Inglioblastoma (GBM), the most aggressive glioma subtype, tumor formationand growth appear to be driven by amplification or overexpression ofgene products involved in growth factor-initiated signal transductionacting in cooperation with genetic alterations disrupting cell-cyclecontrol (Holland E C (2001) Nat Rev Genet 2:120-129). Of the genomicalterations described in GBM, PTEN mutation and/or deletion is the mostcommon, with an estimated frequency of 70-90% (Nutt C, Louis D N (2005)Cancer of the Nervous System (McGraw-Hill, New York), 2nd Ed, pp837-847.). These findings, along with the prognostic value of PTENstatus in GBM cases (Phillips H S, et al. (2006) Cancer Cell 9:157-163),suggest the importance of the phosphoinositide 3-kinase (PI3K)/Aktpathway in promoting highly aggressive glial malignancies, as well asthe opportunities for treatment with PI3K inhibitors possessingblood-brain barrier penetrant properties.

Certain tricyclic PI3K inhibitor compounds of Formula III (below)disclosed in U.S. Pat. No. 8,883,799 have been discovered to possess PI3kinase modulating or inhibitory activity, anti-cancer properties,anti-inflammatory properties and/or immunoregulatory properties.

The Formula III compounds of the U.S. Pat. No. 8,883,799 may be usefulin the treatment of hyperproliferative disorders such as cancer that arecharacterized by the modulation of PI3 kinase function, for example bymutations or overexpression of the proteins. Useful methods forpreparing Formula III are known. However, a need exists for improvedmethods for preparing compounds of Formula III in high yield and purity.

SUMMARY

In some embodiments, the disclosure relates to a process for preparingcompound a Formula III from compound a Formula II in a reaction mixtureaccording to the following reaction scheme:

The process comprises: (i) forming a reaction mixture comprising thecompound Formula II, organoboron-R⁴, the solvent system comprising atleast 5 v/v % water, the base and the catalyst; (ii) reacting thereaction mixture at a temperature of less than 100° C. to form areaction product mixture comprising compound Formula III; and (iii)isolating the compound Formula III, a stereoisomer, geometric isomer,tautomer, or a pharmaceutically acceptable salt thereof, from thereaction product mixture. The catalyst comprises palladium and thereaction mixture comprises less than 0.05 equivalents of catalyst perequivalent of compound Formula II.

Further, X¹ is S, O, N, NR⁶, CR¹, C(R¹)₂, or —C(R¹)₂O—. X² is C, CR² orN. X³ is C, CR³ or N. X⁴ is halogen. A is a 5, 6, or 7-memberedcarbocyclyl or heterocyclyl ring fused to X² and X³, optionallysubstituted with one or more R⁵, R¹⁰ and/or R¹⁵ groups. R¹, R², and R³are independently selected from H, F, Cl, Br, I, —CH₃, —CH₂CH₃,—C(CH₃)₃, —CH₂OH, —CH₂CH₂OH: —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CF₃, —CO₂H,—COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂,—CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl.

Yet further, R⁶ is H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,—(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂ alkylene)(—C₂-C₂₀heterocyclyl), —(C₁-C₁₂ alkylene)-C(═O)—(C₂-C₂₀ heterocyclyl), (C₄-C₁₂alkylene)-(C₆-C₂₀ aryl), and —(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl),where alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl,aryl, and heteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —C(CH₃)₃,—CH₂OH, —CH₂CH₂OH, —(CH₃)₂OH, —CH₂OCH₃, —CN, —CO₂H, —COCH₃, —COC(CH₃)₃,—CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂, —NO₂, —NH₂,—NHCH₃, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂,—N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃,cyclopropyl, cyclobutyl, oxetanyl, morpholino, and1,1-dioxo-thiopyran-4-yl.

Still further, R⁴ is selected from C₆-C₂₀ aryl, C₂-C₂₀ heterocyclyl andC₁-C₂₀ heteroaryl, each of which are optionally substituted with one ormore groups independently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H,—CONH₂, CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃,—OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —NHS(O)₂CH₃,—N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy, morpholinyl,morpholinomethyl, and 4-methylpiperazin-1-yl.

Each R⁵, R¹⁰ and R¹⁵ is independently selected from C₄-C₁₂ alkyl, C₂-C₈alkenyl, C₂-C₈ alkynyl, —(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl),—(C₁-C₁₂ alkylene)-(C₂-C₂₀ heterocyclyl), —(C₁-C₁₂alkylene)-C(O)—(C₂-C₂₀ heterocyclyl), —(C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl),and —(C₄-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl); or two geminal R⁵, R¹⁰and/or R¹⁵ groups form a 3, 4, 5, or 6-membered carbocyclyl orheterocyclyl ring, where alkyl, alkenyl, alkynyl, alkylene, carbocyclyl,heterocyclyl, aryl, and heteroaryl are optionally substituted with oneor more groups independently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CH₂F, —CHF₂,—CF₃, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂,—C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NH—COCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl.

Further, mor is selected from:

wherein mor is optionally substituted with one or more R⁷ groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂, —CN, —CF₃, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH(CH₃)OH, —CH(CH₂CH₃)OH, —CH₂CH(OH)CH₃,—C(CH₃)₂OH, —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂, —CH(CH₂CH₃)F,—C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃, —CON(CH₃)₂, —NO₂,—NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃, —NHCH(CH₃)₂, —NHCH₂CH₂OH,—NHCH₂CH₂OCH₃, —NHCOCH₃, —NHCOCH₂CH₃, —NHCOCH₂OH, —NHS(O)₂CH₃,—N(CH₃)S(O)₂CH₃, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH,—NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃,—S(O)₂NH₂, —S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃.

In some other embodiments, the disclosure relates to a process forpreparing a compound of Formula IIa from a compound of Formula Iaccording to the following reaction scheme:

The process comprises: (i) forming a reaction mixture comprisingcompound Formula I, an organic halide, a solvent system, a phasetransfer catalyst, and a base; (ii) reacting the reaction mixture toform a reaction product mixture comprising compound Formula IIa, astereoisomer, geometric isomer, tautomer, or a pharmaceuticallyacceptable salt thereof; and (iii) isolating compound Formula IIa fromthe reaction product mixture.

Further, the solvent system comprises at least 5 v/v % water. X is ahalide. Each R⁵, R¹⁰ and R¹⁵ is independently selected from H, C₁-C₁₀hydrocarbyl or from C₁-C₅ hydrocarbyl, wherein each hydrocarbyl isoptionally substituted, two geminal R⁵, R¹⁰ and/or R¹⁵ groups are oxo,or two geminal R⁵, R¹⁰ and/or R¹⁵ groups form a 3, 4, 5, 6, or7-membered carbocyclyl or heterocyclyl, wherein the carbocyclyl orheterocyclyl is optionally substituted. Mor is selected from:

wherein mor is optionally substituted with one or more R⁷ groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂, —CN, —CF₃, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH(CH₃)OH, —CH(CH₂CH₃)OH, —CH₂CH(OH)CH₃,—C(CH₃)₂OH, —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂, —CH(CH₂CH₃)F,—C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃, —CON(CH₃)₂, —NO₂,—NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃, —NHCH(CH₃)₂, —NHCH₂CH₂OH:—NHCH₂CH₂OCH₃, —NHCOCH₃, —NHCOCH₂CH₃, —NHCOCH₂OH: —NHS(O)₂CH₃,—N(CH₃)S(O)₂CH₃, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH,—NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃,—S(O)₂NH₂, —S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃. In formula IR²⁰ is —OH or —NHR²¹, R²¹ is as defined for R⁵, and in formula IIa R²⁰is —O— or —NR²¹—.

In some other embodiments, the disclosure relates to a process forpreparing a compound of Formula IIIa from a compound of Formula IIaaccording to the following reaction scheme, wherein compound Formula IIais prepared according to the process described immediately above:

The process comprises: (i) forming a reaction mixture comprisingcompound Formula IIa, organoboron-R⁴, the solvent system comprising atleast 5 v/v % water, the base and the catalyst; (ii) reacting thereaction mixture to form a reaction product mixture comprising compoundFormula IIIa; and (iii) isolating compound Formula IIIa, a stereoisomer,geometric isomer, tautomer, or a pharmaceutically acceptable saltthereof, from the reaction product mixture by solid liquid separationwherein the yield of compound Formula IIIa is at least 75%.

The catalyst comprises palladium and the reaction mixture comprises lessthan 0.05 equivalents of catalyst per equivalent of compound FormulaIIa.

R⁴ is selected from C₆-C₂₀ aryl, C₂-C₂₀ heterocyclyl and C₁-C₂₀heteroaryl, each of which are optionally substituted with one or moregroups independently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H,—CONH₂, CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃,—OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —NHS(O)₂CH₃,—N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy, morpholinyl,morpholinomethyl, and 4-methylpiperazin-yl. Each R⁵, R¹⁰ and R¹⁵ isindependently selected from H, C₁-C₁₀ hydrocarbyl or from C₁-C₅hydrocarbyl, wherein each hydrocarbyl is optionally substituted, twogeminal R⁵, R¹⁰ and/or R¹⁵ groups are oxo, or two geminal R⁵, R¹⁰ and/orR¹⁵ groups form a 3, 4, 5, 6, or 7-membered carbocyclyl or heterocyclyl,wherein the carbocyclyl or heterocyclyl is optionally substituted.

In some other embodiments, the disclosure relates to a method fortreating cancer in a patient wherein the cancer is characterized by theoverexpression of PI3 kinase, the method comprising administering atherapeutically effective amount of a PI3 kinase inhibitor compound ofFormula III as previously defined to a person in need of such treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of the amide impurity of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineduring crystallization from an acetic acid-water solvent system atratios of acetic acid to water of 1:1 v/v %, 4:1 v/v %, 9:1 v/v % and98:2 v/v %.

FIG. 2 shows a plot of the effect of the dose of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineversus time after dosage on pAKT in normal brain tissue, expressed asthe ratio of phosphorylated AKT (pAKT) to total AKT (tAKT).

FIG. 3 shows the in vivo efficacy of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineversus U87 MG Merchant (MG/M) human glioblastoma xenografts in doseescalation studies in subcutaneous tumor-bearing Taconic female NCR nudemice and depicts tumor volume versus dosage regimen (dosage rate andtime of administration).

FIG. 4 shows the effect of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineon the ratio of phosphorylated AKT (pAKT) to total AKT (tAKT) in a U87MG/M human glioblastoma xenograft model after 24 days of continuousdosing at dosage rates of 0.5 mg/kg, 3 mg/kg, 10 mg/kg and 18 mg/kgwherein tumors were excised from animals 1 hour and 4 hours after thelast administered dose on day 24.

FIG. 5 is a ¹H NMR (500 MHz, CDCl₃) spectrum of2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol (compound 5).

FIG. 6 is a ¹³C NMR (125 MHz, CDCl₃) spectrum of2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol (compound 5).

FIG. 7 is a ¹H NMR (500 MHz, CDCl₃) spectrum of2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purine(compound 7).

FIG. 8 is a ¹³C NMR (125 MHz, CDCl₃) spectrum of2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-c]purine(compound 7).

FIG. 9 is a ¹H NMR (500 MHz, CDCl₃) spectrum of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine(pinacolboronate).

FIG. 10 is a ¹³C NMR (125 MHz, CDCl₃) spectrum of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine(pinacolboronate).

FIG. 11 is a ¹H NMR (500 MHz, CDCl₃) spectrum of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine(GDC-0084).

FIG. 12 is a ¹³C NMR (125 MHz, CDCl₃) spectrum of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[4,3-e]purin-2-yl)pyrimidin-2-amine(GDC-0084).

FIG. 13 is a plot of mean±single day plasma concentration vs. timeprofiles of GDC-0084 following a single dose.

FIG. 14 is a plot of ±single day plasma concentration vs. time profilesof GDC-0084 following multiple doses.

FIG. 15 is a GDC-0084 dose proportionality plot of dose (mg) versusC_(max) (μM) for single dose and multiple dose regimens.

FIG. 16 is a GDC-0084 dose proportionality plot of dose (mg) versusAUC₂₄ (μM*hr) for single dose and multiple dose regimens.

FIG. 17 is a plasma GDC-0084 mean single dose concentration versus timelog scale plot.

FIG. 18 is a plasma GDC-0084 mean single dose concentration versus timelinear scale plot.

FIG. 19 is a plasma GDC-0084 mean single dose concentration versus timelog scale plot.

FIG. 20 is a plasma GDC-0084 mean single dose concentration versus timelinear scale plot.

FIG. 21 is a log scale plot of AUC₀₋₂₄ (μM*hr) versus dose (mg) forGDC-0084 for single dose and multiple dose regimens.

FIG. 22 is a log scale plot of Cmax (μM) versus dose (mg) for GDC-0084for single dose and multiple dose regimens.

FIG. 23 is a western blot of mouse brains probed with antibodies againstpAkt, total Akt, pS6, total S6 and actin.

FIG. 24 is a quantitation of pAkt to total Akt and pS6 to total S6 at 1and 6 h post-dose in CD-1 mice.

FIG. 25A depicts images of GDC-0084 mouse brain distribution one hourfollowing oral administration of 15 mg/kg of GDC-0084 in an orthotopicmodel of GS2 glioblastoma intracranial tumors. Localization of thetumors by cresyl violet staining and drug distribution in MALDI MSimages are presented.

FIG. 25B depicts images of GDC-0084 mouse brain distribution one hourfollowing oral administration of 15 mg/kg of GDC-0084 in an orthotopicmodel of U87 glioblastoma intracranial tumors. Localization of thetumors by cresyl violet staining and drug distribution in MALDI MSimages are presented.

FIG. 26A depicts the actual and Gaussian distribution of MALDI imagingsignal intensity of GDC-0084 in orthotopic mouse model U87 intracranialtumors and non-tumor brain regions.

FIG. 26B depicts the actual and Gaussian distribution of MALDI imagingsignal intensity of GDC-0084 in orthotopic mouse model GS2 intracranialtumors and non-tumor brain regions.

FIG. 26C depicts the actual and Gaussian distribution of MALDI imagingsignal intensity of GDC-0084 in the non-tumor regions of the U87 and GS2orthotopic mouse models.

FIG. 26D depicts the actual and Gaussian distribution of signalintensity of GDC-0084 and the actual distribution of pictilisib in theU87 orthotopic GBM model.

FIG. 27A depicts micro-CT images reflecting the tumor size (efficacy) ofGDC-0084 in an U87 orthotopic mouse model following oral administrationof 15 mg/kg GDC-0084 daily for two weeks as compared to treatment with acontrol (GDC-0084 vehicle).

FIG. 27B depicts the tumor volume (in mm³) for mice treated with oraladministration of 15 mg/kg GDC-0084 daily in an U87 orthotopic model ascompared to control mice where the results are presented as themean±S.E. of ten animals.

FIG. 27C depicts representative T-2 weighted MRI images showing theefficacy of GDC-0084 in a GS2 neurosphere tumor mouse model followingoral administration of 15 mg/kg GDC-0084 daily for four weeks ascompared to control animals.

FIG. 27D depicts the tumor volume (in mm³) for mice treated with oraladministration of 15 mg/kg GDC-0084 daily in an U87 orthotopic model ascompared to control mice where the results are presented as themean±S.E. of ten animals.

FIG. 28A depicts a western blot of the PI3K pathway markers pAkt, pS6and p4EBP1 in intracranial GS2 xenografts following oral administrationof 15 mg/kg GDC-0084 daily for four weeks, wherein modulation of thePI3K pathway in the GS2 tumors was assessed by western blot at the endof the 4-week dosing period and at 2 and 8 hours after the finaladministration of 15 mg/kg GDC-0084.

FIG. 28B depicts the quantitation of pAkt/total Akt, p4EBP1/total 4EBP1and pS6/total S6 at 2 and 6 h following the last 15 mg/kg dose ofGDC-0084.

DETAILED DESCRIPTION

Reference will now be made in detail to certain embodiments of thedisclosure, examples of which are illustrated in the accompanyingstructures and formulas. While the disclosure will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the disclosure to those embodiments. Onthe contrary, the disclosure is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present disclosure as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentdisclosure. The present disclosure is in no way limited to the describedmethods and materials. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application prevails.

The present disclosure provides improved processes for preparingtricyclic PI3K inhibitor compounds of Formula III from compounds ofFormula II by Suzuki coupling according to reaction scheme (1):

wherein X¹, X², X³, A, mor, X⁴, organoboron, R⁴, the solvent system, thebase and the catalyst are defined elsewhere herein. The A ring may beoptionally substituted with one or more R⁵, R¹⁰ and/or R¹⁵ groups asdefined elsewhere herein. As compared to known processes, theappropriate selection of at least one process variable from among thecatalyst comprising Pd, the base species, the solvent system, and the/orreaction temperature range, or alternatively the appropriate selectionof 2, 3 or all 4 of these process variables, provides for the improvedyield and/or purity of Formula III, thus enabling the elimination of oneor more process steps and/or purification steps.

The present disclosure further provides processes for forming tricycliccompounds of Formula IIa from bicyclic compounds precursor compounds ofFormula I by annulation through condensation with an alkyl halideaccording to reaction scheme (2):

wherein R⁵, R²⁰, mor, X, the aqueous solvent system, and the base aredefined elsewhere herein. The halo-C₁₋₃ may be optionally substitutedwith one or more R¹⁰ or R¹⁵ groups as defined elsewhere herein. Theformed ring may be optionally substituted with one or more R⁵, R¹⁰and/or R¹³ groups as defined elsewhere herein. As compared to knownprocesses, the appropriate selection of at least one process variablefrom among the solvent system, a phase transfer catalyst, the equivalentratio of two or more of the reactants, and/or the reaction temperaturerange, or alternatively the appropriate selection of 2, 3 or all 4 ofthese process variables, provides for the improved yield and/or purityof compound Formula IIa, thus enabling the elimination of one or moreprocess steps and purification steps.

The present disclosure still further provides improved processes forpreparing tricyclic PI3K inhibitor compounds of Formula IIIa fromcompounds of Formula IIa according to reaction scheme (3):

wherein R⁵, R¹⁰, R¹⁵, R²⁰, mor, X and R⁴ are as defined elsewhereherein. Reaction scheme (3) proceeds generally in accordance withreaction scheme (1).

As further detailed below, the present disclosure still further providesmethod of treatment using the above-noted tricyclic PI3K inhibitorcompounds.

A. Suzuki Coupling

In the Suzuki coupling reaction of reaction scheme (1) and reactionscheme (3), a reaction product mixture comprising compound Formula IIIor IIIa, a stereoisomer, a geometric isomer, a tautomer, or apharmaceutically acceptable salt thereof, is formed from a reactionmixture comprising a compound Formula II or IIa, a solvent systemcomprising water, an organoboron-R⁴, a base and less than 0.05equivalents of a catalyst comprising palladium per equivalent of thecompound Formula II or IIa.

In reaction scheme (1): X¹ is S, O, NR^(a), CR¹, C(R¹)₂ or C(R¹)₂O,wherein R¹ is as further defined below; X² is C, CR² or N, wherein R² isas further defined below; X³ is C, CR³ or N, wherein R³ is as furtherdefined below; X⁴ is a halogen; the dashed lines represent an optionaldouble bond; R⁴ is an optionally substituted C₆-C₂₀ aryl, C₂-C₂₀heterocyclyl or C₁-C₂₀ heteroaryl: A is a 5, 6, or 7-memberedcarbocyclyl or heterocyclyl ring fused to X² and X³, optionallysubstituted with one or more R⁵, R¹⁰ and/or R¹⁵ groups, wherein each R⁵,R¹⁰ and R¹⁵ is independently H, halogen, oxo, hydroxyl, nitro, amino,hydrocarbyl, or substituted hydrocarbyl, two geminal R⁵, R¹⁰ and/or R¹⁵groups are oxo, or two geminal R⁵, R¹⁰ and/or R¹⁵ groups form a 3, 4, 5,6, or 7-membered carbocyclyl or heterocyclyl, wherein the carbocyclyl orheterocyclyl is optionally substituted; and mor is an morpholine ringoptionally substituted with one or more R⁷ groups as defined elsewhereherein.

In some aspects of the disclosure, X¹ is N, NR^(a), CR¹, C(R⁴)₂ orC(R¹)₂O and X³ is C or CR³. In some other aspects, X¹ and X² are N, andX³ is C.

In some aspects of the disclosure, A is an optionally substituted6-membered heterocycle comprising at least one heteroatom selected fromN and O. In some other aspects, A is optionally substituted morpholine.

In some aspects of the disclosure, R⁶ is H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, —(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂alkylene)(—C₂-C₂₀ heterocyclyl), —(C₁-C₁₂ alkylene)-C(═O)—(C₂-C₂₀heterocyclyl), (C₁-C₁₂ alkylene)-(C₆-C₂₀ aryl), and —(C₁-C₁₂alkylene)-(C₁-C₂₀ heteroaryl), where alkyl, alkenyl, alkynyl, alkylene,carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionallysubstituted with one or more groups independently selected from F, Cl,Br, I, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —(CH₃)₂OH, —CH₂OCH₃,—CN, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂,—C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl. In some aspects R⁶ is H orC₁₋₄ alkyl. In still other aspects, R⁶ is H or methyl.

In some aspects, R¹, R², and R³ are independently selected H, F, Cl, Br,I, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃,—CN, —CF₃, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃,—CON(CH₃)₂, —C(CH₃)₂, —CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃,—NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl.

In reaction scheme (3), each R⁵, R¹⁰ and R¹⁵ is independently selectedfrom H, C₁₋₁₀ hydrocarbyl or from C₁₋₅ hydrocarbyl, wherein eachhydrocarbyl is optionally substituted, two geminal R⁵, R¹⁰ and/or R¹⁵groups together are oxo, two geminal R⁵, R¹⁰ and/or R¹⁵ groups togetherform a 3, 4, 5, 6, or 7-membered carbocyclyl or heterocyclyl, whereinthe carbocyclyl or heterocyclyl is optionally substituted. R²⁰ in ring Ais —O— or —NR²¹—, and R²¹ is as defined for R⁵.

In reaction schemes (1) and (3) the organoboron is generally any speciessuitable to achieve the desired yield and/or purity disclosed hereinExamples of organoborons are included in A. Lennox and G Lloyd-Jones,Selection of boron reagents for Suzuki-Miyaum coupling, Chem. Soc. Rev.,2014, 412-443. Non-limiting examples of organoborons include5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2 yl), boronic acid pinacolester, pinacol boronic ester, boronic acids, and organotrifluoroborates.In some particular aspects, the organoboron is5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2 yl).

In some aspects of the disclosure, in reaction schemes (1) and (3), R⁴is independently selected from C₆₋₂₀ aryl, C₂₋₂₀ heterocyclyl and C₁₋₂₀heteroaryl, each of which are optionally substituted with one or moregroups independently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H,—CONH₂, —CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH,—OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃,—NHS(O)₂CH₃, —N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy,morpholinyl, morpholinomethyl, and 4-methylpiperazin-1-yl. In otheraspects, R⁴ is optionally substituted C₆ heteroaryl comprising one ortwo N heteroatoms, or is optionally substituted pyrimidine. In otheraspects, R⁴ is phenyl substituted with one or more groups selected fromF, Cl, Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CN, —CF₃, —CH₂OH, —CO₂H,—CONH₂, —CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH,—OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(═O)NHCH₃, —NHC(═O)NHCH₂CH₃,—NHS(O)₂CH₃, —N(CH₃)C(═O)OC(CH₃)₃, and —S(O)₂CH₃. In still otheraspects, R⁴ is an optionally substituted bicyclic heteroaryl groupselected from 1H-indazole, 1H-indole, indolin-2-one,1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine. In yet other aspects, R⁴ is an optionallysubstituted monocyclic heteroaryl group selected from 2-furanyl,3-furanyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 3-pyrazolyl,4-pyrazolyl, 2-pyrazinyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl,2-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrrolyl, 3-pyrrolyl, 2-thienyl, 3-thienyl, 5-tetrazolyl,1-tetrazolyl, 2-tetrazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,3-triazolyl, and 1-triazolyl. In some aspects, R⁴ is an optionallysubstituted monocyclic heteroaryl group selected from pyridyl,pyrimidinyl and pyrazolyl. In some other aspects, R⁴ is an optionallysubstituted monocyclic pyrimidinyl. In some aspects R⁴ is1H-imidazol-4-yl or 2-aminopyrimidin-yl. In some particular aspects, R4is the optionally substituted moiety R⁴cb depicted below.

Non-limiting examples of the R⁴ moiety include the following whereineach may optionally be substituted and wherein the wavy line indicatesthe site of attachment:

In some aspects, R⁵, R¹⁰ and R¹⁵ are independently selected from C₁₋₁₂alkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, —(C₁₋₁₂ alkylene)-(C₃₋₁₂carbocyclyl), —(C₁₋₁₂ alkylene)-(C₂₋₂₀ heterocyclyl), —(C₁₋₁₂alkylene)-C(O)—(C₂₋₂₀ heterocyclyl), —(C₁₋₁₂ alkylene)-(C₆₋₂₀ aryl), and—(C₁₋₁₂ alkylene)-(C₁₋₂₀ heteroaryl); or two geminal R⁵, R¹⁰ and/or R¹⁵groups form a 3, 4, 5, or 6-membered carbocyclyl or heterocyclyl ring,where alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl,aryl, and heteroaryl are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —C(CH₃)₃,—CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CH₂F, —CHF₂, —CF₃, —CO₂H,—COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂,—C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NH—COCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl.

In some aspects, R⁵, R¹⁰ and R¹³ are independently C₁₋₁₂ alkyloptionally substituted with one or more groups selected from F, Cl, Br,I, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃,—CN, —CH₂F, —CHF₂, —CF₃, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂,—CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂,—NHCOCH₃, —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O,—OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl,oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl. In some aspects, R¹⁰and R¹⁵ are hydrogen and R⁷ is methyl optionally substituted with one ormore groups as defined herein, and in particular may be substituted withone or more substituents selected from F, OH and ═O.

In some other aspects, one or more R⁵, R¹⁰ and/or R¹⁵ groups areindependently selected from H, F, Cl, Br, I, —CH₃, —CH₂CH₃, —C(CH₃)₃,—CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃, —CN, —CH₂F, —CHF₂, —CF₃, —CO₂H,—COCH₃, —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂,—C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃,—N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,—S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl, oxetanyl,morpholino, and 1,1-dioxo-thiopyran-4-yl.

Non-limiting examples of mor include the following where the wavy lineindicates the site of attachment:

In this regard it is to be noted that one or more of the carbon atoms inthe above illustrated rings for mor may be optionally substituted withone or more R⁷ groups independently selected from F, Cl, Br, I, —CH₃,—CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂, —CN, —CF₃,—CH₂OH, —CH₂OCH₃, —CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH(CH₃)OH, —CH(CH₂CH₃)OH,—CH₂CH(OH)CH₃, —C(CH₃)₂OH, —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂,—CH(CH₂CH₃)F, —C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃,—CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃, —NHCH(CH₃)₂,—NHCH₂CH₂OH, —NHCH₂CH₂OCH₃, —NHCOCH₃, —NHCOCH₂CH₃, —NHCOCH₂OH,—NHS(O)₂CH₃, —N(CH₃)S(O)₂CH₃, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH,—NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃,—S(O)₂NH₂, —S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃. In someaspects, mor is mor-a depicted above.

It is to be understood that every embodiment relating to a specificresidue, X¹, X², X³, A, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹⁰, R¹⁵ and mor asdisclosed herein may be combined with any other embodiment relating toanother residue X¹, X², X³, A, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R¹⁰, R¹⁵ andmor as disclosed herein.

The catalyst comprising palladium is generally any such catalystsuitable to achieve the yield and purity disclosed herein. In someaspects, the catalyst comprising palladium is selected fromchloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) (e.g., “Xphos PdG1”, “Xphos PdG2” and “XphosPdG3”); 1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II)complex with dichloromethane (“PdCl₂(dppf).CH₂Cl₂”);Bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II)(“Pd(amphos)Cl₂”);dichlorobis(di-tert-butylphenylphosphine)palladium(II);Dichlorobis(di-tert-butylphenylphosphine)palladium(II)(“PdCl₂[(^(t)Bu₂Ph)P]₂”); PdCl₂(PPh₃)₂;Chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)(Xphos pD G2) of the structure:

Pd(t-Bu)₃; Pd(PPh₃)₄; Pd(OAc)/PPh₃; Cl₂Pd[(Pet₃)]₂; Pd(DIPHOS)₂;Cl₂Pd(Bipy); [PdCl(Ph₂PCH₂PPh₂)]₂; Cl₂Pd[P(o-tol)₃]₂;Pd₂(dba)₃/P(o-tol)₃; Pd₂(dba)/P(furyl)₃; Cl₂Pd[P(furyl)₃]₂;Cl₂Pd(PmePh₂)₂; Cl₂Pd[P(4-F-Ph)₃]₂; Cl₂Pd[P(C₆F₆)₃]₂;Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂; Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂; and encapsulatedcatalysts Pd EnCat™ 30 (palladium acetate, microencapsulated in apolyuria matrix, comprising 0.4 mmol/g Pd), Pd EnCat™ TPP30 (palladiumacetate and triphenylphosphine, microencapsulated in a polyuria matrix,comprising 0.4 mmol/g Pd and 0.3 mmol/g phosphorous), and Pd(II)EnCat™BINAP30 (palladium acetate and BINAP, microencapsulated in a polyuriamatrix, comprising 0.4 mmol/g Pd—see e.g., U.S. Pat. Application Pub.No. 2004/0254066, which is incorporated by reference herein). In someother aspects, the catalyst comprising palladium is selected fromchloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) and1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex withdichloromethane, or ischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II). In some aspects, the catalyst ischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane. In other aspects,the catalyst comprising palladium ischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II).

The equivalent ratio of the catalyst comprising palladium to compoundFormulae II or Ha is typically between about 0.003:1 and about 0.05:1,about 0.003:1 to about 0.03:1 or about 0.004:1 to about 0.02:1. In someparticular aspects, the catalyst ischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)or 1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II) complexwith dichloromethane and the equivalent ratio of the catalyst comprisingpalladium to compound Formulae II or IIa is from about 0.005:1 to about0.04:1, from about 0.005:1 to about 0.03:1, from about 0.01:1 to about0.03:1, or about 0.02:1. In some other particular aspects, the catalystischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)and the equivalent ratio of the catalyst comprising palladium tocompound Formulae II or IIa is from about 0.004:1 to about 0.015:1, fromabout 0.004:1 to about 0.01:1, from about 0.004:1 to about 0.007:1, orabout 0.005:1.

The base may in general be selected from any base that is soluble in thesolvent system and that is suitable to achieve the desired yield andpurity disclosed herein. In some aspects, the base is selected fromK₃PO₄, Cs₂CO₃, K₂CO₃, KOAc, NaOAc, Na₂CO₃ and KOH. In some otheraspects, the base is K₃PO₄. The equivalent ratio of base to compoundFormulae II or IIa is typically at least 1:1, and may be in the range offrom about 1:1 to about 3:1 or about 1.5:1 to about 2.5:1. In aparticular aspect, the ratio may be about 2:1.

In any of the various aspects of the disclosure, the Suzuki couplingsolvent system typically comprises about 5 v/v % water, about 10 v/v %water, about 15 v/v % water, about 20 v/v % water, about 25 v/v % water,or more, and in some aspects may fall within the range of from about 5v/v % to about 25 v/v %, or about 10 v/v % to about 20 v/v %.

The solvent system further comprises at least one co-solvent typicallyselected from non-polar solvents, polar protic solvents and polaraprotic solvents. Suitable polar aprotic solvents include, but are notlimited to, N-methylpyrrolidone, methyl isobutyl ketone, methyl ethylketone, tetrahydrofuran (“THF”), dichloromethane, ethyl acetate,acetone, N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.Suitable polar protic solvents include, but are not limited to,methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol,t-butanol, and acetic acid. Suitable non-polar solvents include dioxane,toluene, hexane, cyclohexane, and diethyl ether.

In some aspects, the solvent system comprises water and at least onepolar aprotic solvent. In some other aspects, the solvent systemconsists essentially of water and a least one polar aprotic solvent. Insome further aspects, the ratio of water to the at least one polaraprotic solvent is from about 1:10 v/v to about 5:1 v/v, from about 1:1v/v to about 1:10 v/v, or from about 1:3 v/v to about 1:7 v/v. In someother aspects, the solvent system consists essentially of water and atleast one polar aprotic solvent.

In some aspects, the solvent system comprises water and THF.

The reaction temperature is typically less than 100° C., and in someaspects may be between about 40° C. and 100° C., from about 40° C. toabout 90° C., from about 40° C. to about 80° C., from about 50° C. toabout 80° C. or from about 55° C. to about 75° C. The reaction time tocompletion is typically from about 4 hours to about 48 hours, from about4 hours to about 36 hours, or from about 4 hours to about 24 hours.

Non-limiting examples of compounds of compound Formulae III and/or IIIainclude the following wherein R¹, R², R³ and R⁴ are as defined elsewhereherein:

In any of the above Formula III compounds, the A ring may be optionallysubstituted with one or more R⁵, R¹⁰ and/or R¹⁵ groups (not shown),which may be independently selected from those options detailedelsewhere herein.

Further non-limiting examples of compounds of Formulae III and IIIainclude wherein R⁴ and R⁵ are as defined elsewhere herein:

Further non-limiting examples of compounds of Formulae III and/or IIIainclude:

In some particular aspects, compound Formulae III and IIIa is:

In some aspects, it has been discovered that precipitation orcrystallization of Formula III and IIIa (collectively referred to asFormula III) from the reaction mixture may be induced by addition ofwater, methanol, ethanol, n-propanol or i-propanol to the reactionmixture to form Formula III crystals or precipitate that may be cleanlyseparated using, for example, solid-liquid separation techniques knownin the art. In some particular aspects the polar protic solvent iswater. In such aspects, the polar protic solvent is added to thereaction mixture to a final concentration of at least 25 v/v %, at least40 v/v %, at least 50 v/v %, at least 60 v/v %, or at least 70 v/v %. Insuch aspects, the volume ratio of the solvent system in the reactionmixture to polar protic solvent added to the reaction product mixture isfrom about 1:5 v/v to about 5:1 v/v, from about 1:3 v/v to about 3:1v/v, from about 1:2 v/v to about 2:1 v/v, from about 1:1.5 v/v to about1.5:1 v/v, or about 1:1 v/v. In any of the various aspects, the polarprotic solvent (e.g., water) may be added to the reaction mixture at thereaction temperature, or at a lower temperature. After addition, thereaction mixture-polar protic solvent admixture may optionally be heldat reaction temperature or from about 10° C. to about 20° C. lower thanthe reaction temperature for from about 0.5 hours to about 8 hours orfrom about 1 hour to about 4 hours. The admixture may then be furthercooled to from about 0° C. to about 10° C. and held for from about 0.5hours to about 8 hours, or from about 1 hour to about 4 hours, tocomplete crystallization of Formula III. In some aspects, in a firststep, the temperature may be reduced to from about 10° C. to about 30°C. and held from about 0.5 hours to about 8 hours or from about 1 hourto about 4 hours and then cooled to 0° C. to about 10° C. in a secondstep held from about 0.5 hours to about 8 hours or from about 1 hour toabout 4 hours. In some other aspects, Formula III seed crystals may beadded to induce crystallization. Formula III solids may be isolated bysolid-liquid separation techniques generally known in the art such asfiltration and centrifugation. Optionally, the collected Formula IIIsolids may be washed with additional polar aprotic solvent.

Formulae II, IIa, III and IIIa may be isolated from the reaction mixtureand purified by various methods generally known in the art. Formula IIIreaction mixtures may comprise some amount of side product (such asImpurities 1 to 5 disclosed, in the examples), unreacted Formula II andpalladium. In some aspects of the disclosure, Formulae II, IIa, III andIIIa reaction mixtures and/or isolated compounds may be purified by oneor more purification methods. The desired product(s) of each step orseries of steps is separated and/or purified (hereinafter separated) tothe desired degree of purity by the techniques common in the art.Formulae II, IIa and III may be isolated from the reaction mixtureand/or purified by various methods including: (i) precipitation orcrystallization such as by evaporation, cooling and/or addition of ananti-solvent in optional further combination with seed crystal addition;(ii) extraction, such as multiphase extraction; (iii) evaporation ordistillation to form a solid residue comprising the Formulae II, IIa orIII; (iv) ultrafiltration; (v) chromatography; (vi) reverse phase HPLC;(vii) sublimation; (viii) chelation; and/or (ix) combinations thereof.Chromatography can involve any number of methods including, for example:reverse-phase and normal phase; size exclusion; ion exchange; high,medium and low pressure liquid chromatography methods and apparatus;small scale analytical; simulated moving bed (SMB) and preparative thinor thick layer chromatography, as well as techniques of small scale thinlayer and flash chromatography.

In chelation methods, Formula ITT is admixed with a reagent selected tobind to or render otherwise separable a desired product, unreactedstarting material, reaction by product, or the like. Such reagentsinclude adsorbents or absorbents such as activated carbon, molecularsieves, ion exchange media, or the like. Alternatively, the reagents canbe acids in the case of a basic material, bases in the case of an acidicmaterial, binding reagents such as antibodies, binding proteins,selective chelators such as crown ethers, liquid/liquid ion extractionreagents (LIX), or the tike. In one chelation purification method,Formula III is processed to remove residual palladium in a methodwhereby Formulae III is admixed with a metal scavenger in a solventsystem in which Formula III is soluble. The temperature of the admixtureis increased to dissolve the compound of Formula III, the solution isheld for a period of time, and the solution is filtered to removechelated palladium. Formula III may then be crystallized from thefiltered solution as described elsewhere herein. In some aspects: (i)the solvent system comprises water and acetic acid, or consistsessentially of water and acetic acid, wherein the volume ratio of aceticacid to w ater is from about 1:1 to about 10:1, from about 1:1 to about5:1 or from about 1:1 to about 3:1, or about 3:1; (ii) the metalscavenger is silica-thiol; and (hi) the dissolution temperature is fromabout 80° C. to about 100° C., the seed crystals are combined with thefiltrate at a temperature of from about 70° C. to about 80° C., and thecrystallization temperature is from about 0° C. to about 10° C.

In any of the various aspects of the disclosure, the yield of compoundFormula III is at least 75%, at least 80% at least 85% or at least 90%.The purity of compound Formula III is at least 97%, at least 97.5%, orat least 98% (area %, as determined by HPLC).

In one or more of the above-noted reactions, the solvent may suitably bewater and THF at a ratio of water to THF of about 1:5 w/w, theorganoboron-R⁴ may suitably be5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine, thecatalyst may suitably be Xphos (e.g., Xphos PdG1, Xphos PdG2 and XphosPdG3) the base may suitably be K₃PO₄, and the reaction temperature maysuitably be about 55° C. to about 75° C. From about 0.8 to about 1.2volumes of water based on the volume of the reaction mixture maysuitably be admixed with the reaction product mixture, and the admixturemay suitably be cooled to from about 10° C. to about 30° C. to formcrystallized or precipitated reaction product.

The Formula III compounds of the disclosure may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of thedisclosure, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present disclosure.

In addition, the present disclosure embraces all geometric andpositional isomers. For example, if a Formula III compound incorporatesa double bond or a fused ring, the cis- and trans-forms, as well asmixtures thereof, are embraced within the scope of the disclosure. Boththe single positional isomers and mixture of positional isomers are alsowithin the scope of the present disclosure.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the disclosure. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present disclosure may be atropisomers(e.g., substituted biaryls) and are considered as part of thisdisclosure. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the disclosure can be separated and isolated by anysuitable method, including: (1) formation of ionic, diastereomeric saltswith chiral compounds and separation by fractional crystallization orother methods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry. Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1) above, diastereomeric salts can be formed by reactionof enantiomerically pure chiral bases such as brucine, quinine,ephedrine, strychnine, α-methyl-β-phenylethylamine (amphetamine), andthe like with asymmetric compounds bearing acidic functionality, such ascarboxylic acid and sulfonic acid. The diastereomeric salts may beinduced to separate by fractional crystallization or ionicchromatography. For separation of the optical isomers of aminocompounds, addition of chiral carboxylic or sulfonic acids, such ascamphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid canresult in formation of the diastereomeric salts.

Alternatively, by method (2) above, the substrate to be resolved isreacted with one enantiomer of a chiral compound to form adiastereomeric pair (E. and Wilen, S. “Stereochemistry of OrganicCompounds”, John Wiley & Sons, Inc., 1994, p. 322). Diastereomericcompounds can be formed by reacting asymmetric compounds withenantiomerically pure chiral derivatizing reagents, such as methylderivatives, followed by separation of the diastereomers and hydrolysisto yield the pure or enriched enantiomer. A method of determiningoptical purity involves making chiral esters of the racemic mixture,such as a methyl ester, for example with (−) methyl chloroformate, inthe presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), and analyzing the ¹H NMR spectrum for the presence ofthe two atropisomeric enantiomers or diastereomers. Stable diastereomersof atropisomeric compounds can be separated and isolated by normal- andreverse-phase chromatography following methods for separation ofatropisomeric naphthyl-isoquinolines (WO 1996/015111). By method (3)above, a racemic mixture of two enantiomers can be separated bychromatography using a chiral stationary phase (“Chiral LiquidChromatography” (1989) W. J. Lough, Ed., Chapman and Hall, New York;Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

The compounds of the present disclosure may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the disclosureembrace both solvated and unsolvated forms.

The compounds of the present disclosure may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe disclosure. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

The present disclosure also embraces isotopically-labeled compounds ofthe present disclosure which are identical to those recited herein, butfor the fact that one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. All isotopes of any particular atom or elementas specified are contemplated within the scope of the compounds of thedisclosure, and their uses. Exemplary isotopes that can be incorporatedinto compounds of the disclosure include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine,such as ²H (D), ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³²P, ³³P,³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certain isotopically-labeled compounds ofthe present disclosure (e.g., those labeled with ³H and ¹⁴C) are usefulin compound and/or substrate tissue distribution assays. Tritiated (³H)and carbon-14 (¹⁴C) isotopes are useful for their ease of preparationand detectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸Fare useful for positron emission tomography (PET) studies to examinesubstrate receptor occupancy. Isotopically labeled compounds of thepresent disclosure can generally be prepared by following proceduresanalogous to those disclosed in the Schemes and/or in the Examplesherein by substituting an isotopically labeled reagent for anon-isotopically labeled reagent.

B. Annulation

In the annulation (ring forming) reaction of reaction scheme (2): eachR⁵, each R¹⁰ and each R¹⁵ is independently selected from H, C₁-C₁₀hydrocarbyl or from C₁-C₅ hydrocarbyl, wherein each hydrocarbyl isoptionally substituted; two geminal R⁵ groups, R¹⁰ groups and/or R¹⁵groups together are oxo or together form a 3, 4, 5, 6, or 7-memberedcarbocyclyl or heterocyclyl, wherein the carbocyclyl or heterocyclyl isoptionally substituted. R²⁰ is —OH or —NHR²¹; R²¹ is as defined for R⁵;and R²⁰ in the ring formed in Formula IIa is —O— or —NR²¹—. Mor is anoptionally substituted morpholine ring. X is halogen (e.g., Br, I orCl). In some aspects of the present disclosure, compounds of Formula IIaare formed from bicyclic compounds precursor compounds of Formula I byannulation through condensation with a nucleophile derived from 1,2ethane diol and epoxide, such as an organic halide. In any of thevarious such reactions, a reaction mixture is formed comprising acompound Formula I, halo-alkyl, a solvent system, a phase transfercatalyst, and a base. The reaction mixture is reacted at elevatedtemperature to form a reaction product mixture comprising compoundFormula IIa, a stereoisomer, geometric isomer, tautomer, or apharmaceutically acceptable salt thereof, and compound Formula IIa isthen isolated from the reaction product mixture. In some aspects of thepresent disclosure, compound Formula IIa may be purified in one or morepurification steps.

In some aspects of the disclosure, the solvent system comprises at leastone polar protic solvent. Examples of such solvents include, withoutlimitation, water, methanol, ethanol, n-propanol, i-propanol, n-butanol,i-butanol, t-butanol, and acetic acid (“HOAc”). In such aspects, thesolvent system comprises at least 5 v/v % water, at least 25 v/v %water, at least 50 v/v % water, at least 75 v/v % water, at least 90 v/v% water, or consists essentially of water.

In some aspects of the disclosure, the solvent system comprises at leastone polar aprotic solvent. Examples of such solvents include, withoutlimitation, dichloromethane, tetrahydrofuran (“THF”), ethyl acetate,acetone, N—N-dimethylformamide (“DMF”), acetonitrile (“MeCN”),dimethylsulfoxide, N-methylpyrrolidone (“NMP”), methyl isobutyl ketoneand methyl ethyl ketone. In such aspects, the solvent comprises DMF orpredominantly comprises DMF.

In yet other aspects of the disclosure, the solvent system comprises atleast one polar protic solvent and at least one polar aprotic solvent ata ratio of total polar protic solvent to total polar aprotic solvent offrom about 1:10 to about 10:1, from about 1:5 to about 5:1, from about1:10 to about 1:1, from about 1:5 to about 1:1, from about 10:0 to about1:1, or from about 5:1 to about 1:1. In some such aspects, the solventsystem comprises water and THF or water and DMF.

Total solvent loading, in terms of volumes based on Formula I, is about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 15 or about 20, and ranges thereof, such as from about 2 toabout 20, from about 2 to about 15, from about 2 to about 10, from about5 to about 15, from about 2 to about 7, from about 3 to about 7, or fromabout 3 to about 5.

The nucleophile for annulation by condensation reaction may be anucleophile derived from (i) 1,2-ethanediol or a mesylate, tosylate ortriflate derivative thereof or (n) an epoxide. In some aspects, thenucleophile is a halide or pseudohalide, such as an organic halide. Insome aspects, the organic halide is a halogenated alkyl. In someparticular aspects, the halide is a Cl, Br or I halogenated C₃ to C₃alkyl. In some aspects, the organic halide is optionally substituted. Insome aspects the alkyl is di-substituted with halogen at the terminalends. An example of one such organic halide is 1,2-dibromoethane. Theequivalent ratio of organic halide to compound Formula I is greater than2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about 4.5:1 orabout 5:1, and ranges thereof, such as between 2:1 and about 5:1,between 2:1 and about 4:1, from about 2.5:1 to about 4:1, from about2.5:1 to about 3.5:1, or about 3:1. The organic halide may be combinedwith the reaction mixture in one or more addition steps.

In some aspects of the present disclosure, the reaction mixturecomprises a phase transfer catalyst. In some such aspects, the phasetransfer catalyst may be selected from a quaternary ammonium salt and aphosphonium salt. Examples of such phase transfer catalysts include,without limitation, tetra-n-butylammonium bromide (“TBAB”),benzyltrimethylammonium chloride, benzyltriethylammonium chloride,methyltricaprylammonium chloride, methyltributylammonium chloride, andmethyltrioctyl ammonium chloride. In particular aspects, the phasetransfer catalyst is TBAB. The equivalent ratio of phase transfercatalyst to compound Formula I is about 0.1:1, about 0.2:1, about 0.3:1,about 0.4:1, about 0.5:1 or about 0.6:1 and ranges thereof, such fromabout 0.1:1 to about 0.6:1, from about 0.2:1 to about 0.5:1, from about0.2:1 to about 0.4:1, or about 0.3:1.

In any of the various annulation aspects of the present disclosure, thereaction mixture comprises a base. Examples of suitable bases include,without limitation, KOH, NaOH, K₃PO₄, K₂CO₃, NaHCO₃ and Cs₂CO₃. In someaspects, the base is KOH, NaOH or K₃PO₄, and in other aspects the baseis KOH. The equivalent ratio of the base to compound Formula I isgreater than 2:1, about 2.5:1, about 3:1, about 3.5:1, about 4:1, about4.5:1 or about 5:1, and ranges thereof, such as between 2:1 and about5:1, between 2:1 and about 4:1, from about 2.5:1 to about 4:1, fromabout 2.5:1 to about 3.5:1, or about 3:1. In some aspects, the base andthe organic halide are present in about equimolar amounts. The base maybe combined with the reaction mixture in one or more addition steps.

The reaction temperature is suitably from about 40° C. to about 90° C.,from about 40° C. to about 70° C., from about 40° C. to about 60° C., orabout 50° C. Based on experimental evidence to date, it has beendiscovered that the purity profile of Formula IIa varies inversely withreaction temperature, such that lower reaction temperatures provide forimproved purity profiles as measured by HPLC as compared with higherreaction temperatures. The reaction time required to achieve sufficientconversion varies with the quantitative and qualitative characteristicsof the reaction mixture and the reaction temperature, and is typicallyabout 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20hours, about 24 hours or about 28 hours, and ranges thereof, such asabout 4 hours to about 28 hours, about 4 hours to about 20 hours orabout 4 hours to about 12 hours.

Compound Formula IIa may be isolated from the reaction mixture byvarious methods including precipitation, crystallization and extraction.In an example of a first extraction method, the reaction mixture may becooled, such as to room temperature, diluted with water (e.g., 5 to 15volumes of water) and held with agitation (e.g., 5 to 15 hours). FormulaIIa may then be extracted from the diluted reaction mixture into anorganic solvent (e.g., 1 to 3 volumes of a suitable solvent such aspolar aprotic solvent such as ethyl acetate) followed by isolation ofthe organic solvent from the reaction mixture by phase separation.Multiple such extractions are within the scope of the first method. Theorganic phase may suitably be washed with NaCl and concentrated (e.g.,by distillation) to yield crude solid Formula IIa. Crude Formula IIa mayoptionally be crystallized such as by dissolution in a suitable solventat elevated temperature (e.g., 65° C. in isopropanol) followed bycrystallization by cooling. In an example of a second extraction method,water (e.g., 5 to 10 volumes) and an organic solvent (e.g., 2 to 6volumes ethyl acetate) may be added to a cooled reaction mixturefollowed by phase separation to remove the organic layer. In someaspects, additional water (e.g. 2 to 6 volumes) and organic solvent(e.g., 2 to 6 volumes ethyl acetate) may be added to the aqueous phasefollowed by phase separation to remove the organic layer. The combinedorganic layers may be concentrated (e.g., by distillation) to yieldsolid crude Formula IIa. Formula IIa may optionally be combined with asolvent (e.g., isopropanol) followed by a second concentration step.Crude Formula IIa may optionally be crystallized such as by dissolutionin a suitable solvent at elevated temperature (e.g., 65° C. inisopropanol) followed by crystallization by cooling. In aspects of thedisclosure where the reaction mixture solvent system comprises water, analcohol (e.g., ethanol) may be added to the reaction product mixture andthe temperature of the admixture may be reduced to inducecrystallization of compound Formula IIa. In some optional aspects, seedcrystals of compound Formula IIa may be added to the admixture. In suchaspects, the volume ratio of the reaction mixture solvent system toalcohol is from about 1:5 v/v to about 5:1 v/v, from about 1:3 v/v toabout 3:1 v/v, from about 1:2 v/v to about 2:1 v/v, from about 1:1 v/vto about 1:2 v/v, or about 1:1.3 v/v Crystallized compound Formula IIamay be isolated by solid liquid separation techniques known in the art.

In any of the various aspects, the yield of compound Formula IIa is atleast 65% at least 70% or at least 75%. The purity of compound FormulaIIa is at least 97%, at least 97.5%, at least 98%, at least 98.5% or atleast 99% (area % as determined by HPLC).

Non-limiting examples of reactions within the scope of the presentdisclosure include:

In one or more of the above-noted reactions, the solvent may suitably bewater, the base may suitably be KOH, the phase transfer catalyst maysuitably be tetra-n-butylammonium bromide, and the reaction temperaturemay suitably be about 50° C. Ethanol may suitably be admixed with thereaction product mixture and the admixture may suitably be cooled tofrom about 0° C. to about 10° C. in the presence of reaction productseed crystals to form crystallized reaction product.

C. Exemplary Embodiments

In a first exemplary embodiment of the present disclosure, the compoundof Formula IIIat (GDC-0084):

or a salt thereof, is prepared by a process comprising contacting acompound of Formula IIa:

or a salt thereof, wherein X is chloro or bromo, in a solvent systemcomprising a least 5% v/v % water and at a reaction temperature of lessthan 100° C. with an organoboron-pyrimidin-2-amine in the presence of abase and less than 0.05 equivalents of a Suzuki coupling palladiumcatalyst per equivalent of compound Formula IIa.

In one aspect of the first exemplary embodiment, the solvent systemcomprises water and tetrahydrofuran, wherein the ratio of water totetrahydrofuran is from about 1:3 v/v to about 1:7 v/v, or about 1:5v/v. In some other aspects of the first exemplary embodiment, theorganoboron-pyrimidin-2-amine is5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine. In yetother aspects of the first exemplary embodiment, the base is K₃PO₄ andthe equivalent ratio of the base to compound Formula IIa is from about1:1 to about 3:1, or about 2:1. In still other aspects of the firstembodiment, the catalyst ischloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)and the equivalent ratio of the catalyst comprising palladium tocompound Formula IIa is from about 0.004:1 to about 0.007:1, or about0.005:1. In still other aspects of the first embodiment, the catalyst is1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II) complex withdichloromethane and the equivalent ratio of the catalyst comprisingpalladium to compound Formula IIa is from about 0.01:1 to about 0.03:1,or about 0.02:1. In yet other aspects of the first embodiment, thereaction temperature is from about 55° C. to about 75° C. In any of thevarious aspects of the first embodiment, water is added to the reactionproduct mixture to form an admixture comprising greater than 25 v/v %water to form a precipitate or crystalline compound Formula IIIa, whichmay be isolated from the reaction product mixture. In some such aspects,the volume ratio of the solvent system to added water is from about1:1.5 v/v to about 1.5:1 v/v, or about 1:1 v/v.

In a second exemplary embodiment of the present disclosure, the compoundof Formula IIa:

or a salt thereof, is prepared by a process comprising: (a) forming areaction mixture comprising a solvent system comprising at least 5 v/v %water, 1,2-dibromoethane, a phase transfer catalyst, a base, andcompound Formula I:

or a salt thereof, wherein X is bromo, chloro or iodo; and, (b) reactingthe reaction mixture to form a reaction product mixture comprisingcompound Formula IIa.

In one aspect of the second exemplary embodiment, the solvent systemcomprises at least 90 v/v % water or consists essentially of water. Insome other aspects of the second exemplary embodiment, the base is KOHand the phase transfer catalyst is tetra-n-butylammonium bromide. In yetother aspects, the mole ratio of 1,2-dibromoethane to compound Formula Iis between about 2:1 and about 4:1, or about 3:1, and the mole ratio of1,2-dibromoethane to the base is about 1:1. In other aspects, thereaction temperature is from about 40° C. to about 60° C., such as about50° C. In other aspects, ethanol is admixed with the reaction productmixture followed by cooling of the admixture to form crystallizedcompound Formula IIa, wherein the volume ratio of the solvent system toethanol is from about 1:1 v/v to about 1:2 v/v, or about 1:1.3 v/v %. Inyet other aspects, compound Formula IIa seed crystals are combined withthe ethanol-reaction product mixture.

D. Methods of Treatment

The Formula III compounds of the invention may be administered by anyroute appropriate to the condition to be treated. Suitable routesinclude oral, parenteral (including subcutaneous, intramuscular,intravenous, intraarterial, intradermal, intrathecal and epidural),transdermal, rectal, nasal, topical (including buccal and sublingual),vaginal, intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with e.g. the conditionof the recipient. Where the compound is administered orally, it may beformulated as a pill, capsule, tablet, etc. with a pharmaceuticallyacceptable carrier or excipient. Where the compound is administeredparenterally, it may be formulated with a pharmaceutically acceptableparenteral vehicle and in a unit dosage injectable form, as detailedbelow.

In one embodiment, the composition comprising a compound of Formula IIIor salt thereof is formulated as a solid dosage form for oraladministration. Solid dosage forms for oral administration includecapsules, tablets, pills, powders, and granules. In certain embodiments,the solid oral dosage form comprising a compound of Formula III or asalt thereof further comprises one or more of (i) an inert,pharmaceutically acceptable excipient or carrier, such as sodium citrateor dicalcium phosphate, and (ii) filler or extender such as starches,lactose, sucrose, glucose, mannitol, or silicic acid, (iii) binders suchas carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose or acacia, (iv) humectants such as glycerol, (v) disintegratingagent such as agar, calcium carbonate, potato or tapioca starch, alginicacid, certain silicates or sodium carbonate, (vi) solution retardingagents such as paraffin, (vii) absorption accelerators such asquaternary ammonium salts, (viii) a wetting agent such as cetyl alcoholor glycerol monostearate, (ix) absorbent such as kaolin or bentoniteclay, and (x) lubricant such as talc, calcium stearate, magnesiumstearate, polyethylene glycols or sodium lauryl sulfate. In certainembodiments, the solid oral dosage form is formulated as capsules,tablets or pills. In certain embodiments, the solid oral dosage formfurther comprises buffering agents. In certain embodiments, suchcompositions for solid oral dosage forms may be formulated as fillers insoft and hard-filled gelatin capsules comprising one or more excipientssuch as lactose or milk sugar, polyethylene glycols and the like.

In certain embodiments, tablets, dragees, capsules, pills and granulesof the compositions comprising a compound of Formula III or salt thereofoptionally comprise coatings or shells such as enteric coatings. Theymay optionally comprise opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions includepolymeric substances and waxes, which may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

In another embodiment, a composition comprises micro-encapsulatedcompound of Formula III or salt thereof, and optionally, furthercomprises one or more excipients.

In another embodiment, compositions comprise liquid dosage formulationscomprising a compound of Formula III or salt thereof for oraladministration, and optionally further comprise one or more ofpharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In certain embodiments, the liquiddosage form optionally, further comprise one or more of an inert diluentsuch as water or other solvent, a solubilizing agent, and an emulsifiersuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols or fatty acid esters ofsorbitan, and mixtures thereof. In certain embodiments, liquid oralcompositions optionally further comprise one or more adjuvant, such as awetting agent, a suspending agent, a sweetening agent, a flavoring agentand a perfuming agent.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use.

In order to prolong the effect of a compound of Formula III, it is oftendesirable to slow the absorption of the compound from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor w atersolubility. The rate of absorption of the compound then depends upon itsrate of dissolution that, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of a parenterallyadministered compound form is accomplished by dissolving or suspendingthe compound in an oil vehicle. Injectable depot forms are made byforming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

In certain embodiments, the composition for rectal or vaginaladministration are formulated as suppositories which can be prepared bymixing a compound of Formula III or a salt thereof with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, for example those which are solid atambient temperature but liquid at body temperature and therefore melt inthe rectum or vaginal cavity and release the compound of Formula III.

Example dosage forms for topical or transdermal administration of acompound of Formula III include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants or patches. The compound ofFormula III or a salt thereof is admixed under sterile conditions with apharmaceutically acceptable carrier, and optionally preservatives orbuffers. Additional formulation examples include an ophthalmicformulation, ear drops, eye drops, transdermal patches. Transdermaldosage forms can be made by dissolving or dispensing the compound ofFormula III or a salt thereof in medium, for example ethanol ordimethylsulfoxide. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate can be controlled byeither providing a rate controlling membrane or by dispersing thecompound in a polymer matrix or gel.

Nasal aerosol or inhalation formulations of a compound of Formula III ora salt thereof may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promotors to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

In certain embodiments, pharmaceutical compositions may be administeredwith or without food. In certain embodiments, pharmaceuticallyacceptable compositions are administered without food. In certainembodiments, pharmaceutically acceptable compositions of this inventionare administered with food.

Specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including age, body weight, generalhealth, sex, diet, time of administration, rate of excretion, drugcombination, the judgment of the treating physician, and the severity ofthe particular disease being treated. The amount of a provided compoundof Formula III or salt thereof in the composition will also depend uponthe particular compound in the composition.

In one embodiment, the therapeutically effective amount of the compoundof the invention administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, alternatively about 0.1 to 20 mg/kg of patientbody weight per day, with the typical initial range of compound usedbeing about 0.1 to 15 mg/kg/day, about 0.1 to 10 mg/kg/day, about 0.1 to5 mg/kg/day, about 0.1 to 3 mg/kg/day, about 0.3 to 1.5 mg/kg/day, orabout 0.4 to 1 mg/kg/day. In another embodiment, oral unit dosage forms,such as tablets and capsules, contain from about 5 to about 100 mg,about 10 to 75 mg, about 25 to 75 mg, or about 25 to 50 mg of thecompound of the invention.

An example tablet oral dosage form comprises about 2 mg, about 5 mg,about 25 mg, about 50 mg, about 100 mg, about 250 mg or about 500 mg ofa compound of Formula III or salt thereof, and further comprises about5-30 mg anhydrous lactose, about 5-40 mg sodium croscarmellose, about5-30 mg polyvinylpyrrolidone (PVP) K30 and about 1-10 mg magnesiumstearate. The process of formulating the tablet comprises mixing thepowdered ingredients together and further mixing with a solution of thePVP. The resulting composition can be dried, granulated, mixed with themagnesium stearate and compressed to tablet form using conventionalequipment. An example of an aerosol formulation can be prepared bydissolving about 2-500 mg of a compound of Formula III or salt thereof,in a suitable buffer solution, e.g. a phosphate buffer, and adding atonicifier, e.g. a salt such sodium chloride, if desired. The solutionmay be filtered, e.g. using a 0.2 micron filter, to remove impuritiesand contaminants.

A dose to treat human patients may range from about 10 mg to about 1000mg, from about 10 mg to about 500 mg, from about 10 mg to about 100 mg,from about 25 mg to about 100 mg, or from about 25 mg to about 75 mg ofFormula III compound. A typical dose may be about 100 mg to about 300 mgof the compound. A dose may be administered once a day (QID or QD),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

In some embodiments, a total of from about 0.2 mg/kg/day to about 1.5mg/kg/day, from about 0.3 mg/kg/day to about 1 mg/kg/day, or from about0.4 mg/kg/day to about 0.75 mg/kg/day of compound Formula III isadministered once daily or in a twice daily dosage regimen. In some suchembodiments, under such a dosage regimen the following pharmacokineticresults are achieved for a single dose on the first day of a dosagecycle. A T_(1/2) (hr) of from about 10 to about 24 hours, from about 12to about 22 hours, or from about 15 to about 20 hours. A T_(max) (hr) offrom about 1 to about 8 hours, from about 2 to about 6 hours, from about2 to about 4 hours, or from about 2 to about 3 hours. A C_(max) (μM) offrom about 0.01 to about 0.5 μM, from about 0.05 to about 0.4 μM, orfrom about 0.1 to about 0.3 μM. An AUC_(inf) (μM*hr) of from about 0.2to about 10 μM*hr, from about 0.5 to about 10 μM*hr, from about 1 toabout 8 μM*hr, or from about 2 to about 6 μM*hr. An AUC₀₋₂₄ (μM*hr) offrom about 0.1 to about 10 μM*hr, from about 0.5 to about 5 μM*hr, fromabout 1 to about 5 μM*hr, or from about 2 to about 4 μM*hr. In someother such embodiments, the following pharmacokinetic results areachieved for a single dose after the 15^(th) day of a 2 mg to 30 mg perday dosage regimen or for a single does after the 8^(th) day of a 45 mgto 65 mg per day dosage regimen. A T_(max) (hr) of from about 1 to about5 hours, from about 1 to about 3 hours, or from about 2 to about 4hours. A C_(max) (μM) of from about 0.03 to about 1 μM, from about 0.1to about 1 μM, from about 0.3 to about 0.8 μM, or from about 0.3 toabout 0.6 μM. A C_(min) (μM) of from about 0.005 to about 0.5 μM, fromabout 0.01 to about 0.4 μM, from about 0.05 to about 0.3 μM, or fromabout 0.1 to about 0.3 μM. An AUC₀₋₂₄ (μM*hr) of from about 0.1 to about15 μM*hr, from about 0.5 to about 15 μM*hr, from about 3 to about 15μM*hr, or from about 5 to about 10 μM*hr. An accumulation ratio of fromabout 1 to about 4 or from about 1.5 to about 3. As used herein, T_(1/2)refers to terminal half-life; T_(max) refers to time to maximum plasmaconcentration; C_(max) refers to maximum observed plasma concentration;AUC_(inf) refers to area under the concentration-time curve from Time 0to infinity; AUC₀₋₂₄ refers to refers to area under theconcentration-time curve from Time 0 to 24 hours; C_(min) refers tominimum concentration; and Accumulation Ratio refers toAUC_(0-24 hr multiple dose)/AUC_(0-24 hr single dose). In someparticular embodiments, compound Formula III is GDC-0084 (FormulaIIIat).

Formula III compounds may be useful for treating conditions of the brainand central nervous system which require transport across theblood-brain barrier. Certain Formula III compounds, such as compoundFormula IIIat (GDC-0084) disclosed elsewhere herein, have favorablepenetrant properties across the blood-brain barrier for delivery to thebrain. Disorders of the brain which may be effectively treated withFormula III compounds include metastatic and primary brain tumors, suchas glioma (glioblastoma multiforme) and melanoma.

The compounds of Formula III or salts thereof may be employed alone orin combination with other agents for treatment. For example, the secondagent of the pharmaceutical combination formulation or dosing regimenmay have complementary activities to the compound of Formula III suchthat they do not adversely affect each other. The compounds may beadministered together in a unitary pharmaceutical composition orseparately. In one embodiment a compound or a pharmaceuticallyacceptable salt can be co-administered with a cytotoxic agent to treatproliferative diseases and cancer.

The term “co-administering” refers to either simultaneousadministration, or any manner of separate sequential administration, ofa compound of Formula III or a salt thereof, and a further activepharmaceutical ingredient or ingredients, including cytotoxic agents. Ifthe administration is not simultaneous, the compounds are administeredin a close time proximity to each other. Furthermore, it does not matterif the compounds are administered in the same dosage form, e.g. onecompound may be administered topically and another compound may beadministered orally.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of Formula III,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. In certainembodiments, compositions of this invention are formulated such that adosage of between 0.01-100 mg/kg body weight/day of an inventive can beadministered.

Typically, any agent that has activity against a disease or conditionbeing treated may be co-administered. Examples of such agents can befound in Cancer Principles and Practice of Oncology by V. T. Devita andS. Hellman (editors), 6^(th) edition (Feb. 15, 2001), LippincottWilliams & Wilkins Publishers. A person of ordinary skill in the artwould be able to discern which combinations of agents would be usefulbased on the particular characteristics of the drugs and the diseaseinvolved.

In one embodiment, the treatment method includes the co-administrationof a compound of Formula III or a pharmaceutically acceptable saltthereof and at least one cytotoxic agent. The term “cytotoxic agent” asused herein refers to a substance that inhibits or prevents a cellularfunction and/or causes cell death or destruction. Cytotoxic agentsinclude, but are not limited to, radioactive isotopes (e.g., At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu); chemotherapeutic agents; growth inhibitory agents;enzymes and fragments thereof such as nucleolytic enzymes; and toxinssuch as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, including fragments and/orvariants thereof.

In some embodiments, the PI3 kinase inhibitors of the present disclosureare administered to a patient with an additional therapeutic agentselected from a chemotherapeutic agent, an anti-angigenesis therapeuticagent, an anti-inflammatory agent, an immunomodulatory agent, aneurotropic factor, an agent for treating cardiovascular disease, anagent for treating liver disease, an anti-viral agent, an agent fortreating blood disorders, an agent for treating diabetes, and an agentfor treating immunodeficiency disorders. In some such embodiments, theadditional therapeutic agent is bevacizumab. In some other suchembodiments, the additional therapeutic agent is temozolomide.

Exemplary cytotoxic agents can be selected from anti-microtubule agents,platinum coordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A;inhibitors of fatty acid biosynthesis; cell cycle signaling inhibitors;HDAC inhibitors, proteasome inhibitors; and inhibitors of cancermetabolism.

“Chemotherapeutic agent” includes chemical compounds useful in thetreatment of cancer. Examples of chemotherapeutic agents includeerlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®,Millennium Pharm.), disulfiram, epigallocatechin gallate,salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol,lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca),sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis),oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin,Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016,Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, BayerLabs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents suchas thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingtopotecan and irinotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);adrenocorticosteroids (including prednisone and prednisolone);cyproterone acetate; 5α-reductases including finasteride anddutasteride); vorinostat, romidepsin, panobmostat, valproic acid,mocetinostat dolastatin; aldesleukin, talc duocarmycin (including thesynthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, chlorophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin γ1I andcalicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR®(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®(vinorelbine); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Chemotherapeutic agent also includes (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators (SERMs), including, forexample, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON® (toremifine citrate); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprohde and goserelin; buserelin, tripterelin,medroxyprogesterone acetate, diethylstilbestrol, premarin,fluoxymesterone, all transretionic acid, fenretinide, as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceuticallyacceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes antibodies such as alemtuzumab(Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®,Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®,Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech),trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), andthe antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Additional humanized monoclonal antibodies with therapeutic potential asagents in combination with the compounds of the invention include:apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine,cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusttuzumab,cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab,felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin,ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab,motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab,numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab,peefusituzumab, pectuzumab, pexehzumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siphzumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab,ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695,Wyeth Research and Abbott Laboratories) which is a recombinantexclusively human-sequence, full-length IgG₁ λ antibody geneticallymodified to recognize interleukin-12 p40 protein.

Chemotherapeutic agent also includes “EGFR inhibitors,” which refers tocompounds that bind to or otherwise interact directly with EGFR andprevent or reduce its signaling activity, and is alternatively referredto as an “EGFR antagonist.” Examples of such agents include antibodiesand small molecules that bind to EGFR. Examples of antibodies which bindto EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No.4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targetedantibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat.No. 5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAh 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659,439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451. WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

Chemotherapeutic agents also include “tyrosine kinase inhibitors”including the EGFR-targeted drugs noted in the preceding paragraph;small molecule HER2 tyrosine kinase inhibitor such as TAK165 availablefrom Takeda; CP-724,714, an oral selective inhibitor of the ErbB2receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such asEKB-569 (available from Wyeth) which preferentially binds EGFR butinhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016;available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinaseinhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1signaling; non-HER targeted TK inhibitors such as imatinib mesylate(GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosinekinase inhibitors such as sunitinib (SUTENT®, available from Pfizer);VEGF receptor tyrosine kinase inhibitors such as vatalanib(PTK787/ZK222584, available from Novartis/Schering AG); MAPKextracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinibmesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474(AstraZeneca), PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),rapamycin (sirolimus, RAPAMUNE®); or as described in any of thefollowing patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016(American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983(Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (WarnerLambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons,colchicine, metoprine, cyclosporine, amphotericin, metronidazole,alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide,asparaginase, BCG live, bevacuzimab, bexarotene, cladribine,clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa,elotinib, filgrastim, histrelin acetate, ibritumomab, interferonalfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna,methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, plicamycin, porfimer sodium, quinacrine,rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene,tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, andpharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate and fluprednidene acetate; immuneselective anti-inflammatory peptides (ImSAIDs) such asphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such asazathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumornecrosis factor alpha (TNFα) blockers such as etanercept (Enbrel),infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra(Kineret), T cell costimulation blockers such as abatacept (Orencia),Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha(IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such asrhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secretedhomotrimeric LTa3 and membrane bound heterotrimer LTa1/β2 blockers suchas Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu); miscellaneous investigational agents such asthioplatin. PS-341, phenylbutyrate, ET-18-OCH₃, or farnesyl transferaseinhibitors (L-739749, L-744832); polyphenols such as quercetin,resveratrol, piceatannol, epigallocatechine gallate, theaflavins,flavanols, procyanidins, betulinic acid and derivatives thereof;autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin);podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®);bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R);vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g.celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; farnesyltransferaseinhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatorydrugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDsinclude non-selective inhibitors of the enzyme cyclooxygenase. Specificexamples of NSAIDs include aspirin, propionic acid derivatives such asibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen,acetic acid derivatives such as indomethacin, sulindac, etodolac,diclofenac, enolic acid derivatives such as piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivativessuch as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib,parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicatedfor the symptomatic relief of conditions such as rheumatoid arthritis,osteoarthritis, inflammatory arthropathies, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic.

In certain embodiments, chemotherapeutic agents include, but are notlimited to, doxorubicin, dexamethasone, vincristine, cyclophosphamide,fluorouracil, topotecan, interferons, platinum derivatives, taxanes(e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine),anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g.,etoposide), cisplatin, an mTOR inhibitor (e.g., a rapamycin),methotrexate, actinomycin D, dolastatin 10, colchicine, trimetrexate,metoprine, cyclosporine, daunorubicin, teniposide, amphotericin,alkylating agents (e.g., chlorambucil), 5-fluorouracil, campthothecin,cisplatin, metronidazole, and imatinib mesylate, among others. In otherembodiments, a compound of the present invention is administered incombination with a biologic agent, such as bevacizumab or panitumumab.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with an antiproliferative or chemotherapeutic agent selectedfrom any one or more of abarelix, aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, asparaginase, azacitidine, BCG live, bevacuzimab,fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, calusterone,capecitabine, camptothecin, carboplatin, carmustine, cetuximab,chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine,dactinomycin, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane,docetaxel, doxorubicin (neutral), doxorubicin hydrochloride,dromostanolone propionate, epirubicin, epoetin alfa, elotinib,estramustine, etoposide phosphate, etoposide, exemestane, filgrastim,floxuridine, fludarabine, fulvestrant, gefitinib, gemcitabine,gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea,ibritumomab, idarubicin, ifosfamide, imatinib mesylate, interferonalfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole,leucovorin, leuprolide acetate, levamisole, lomustine, megestrolacetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate,methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone,nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, pentostatin, pipobroman, plicamycin, porfimersodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim,sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen,temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG,thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin,ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine,zoledronate, or zoledronic acid.

E. Definitions

The term “hydrocarbyl” as used herein describes organic compounds orradicals consisting exclusively of the elements carbon and hydrogen.These moieties include, without limitation, alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with ahetero atom such as nitrogen, oxygen, silicon, phosphorous, boron,sulfur, or a halogen atom. These substituents include, but are notlimited to, halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy,hydroxy, keto, acyl, acyloxy, nitro, tertiary amino, amido, nitro,cyano, thio, sulfinate, sulfonamide, ketals, acetals, esters and ethers.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁₋₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁₋₈), or oneto six carbon atoms (C₁₋₆). Examples of alkyl groups include, but arenot limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl,3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl,2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl,3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl,2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl,1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁₋₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁₋₈), or one to six carbon atoms (C₁₋₆). Examples of alkylene groupsinclude, but are not limited to, methylene, ethylene, propylene, and thelike.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂₋₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl, allyl, and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to twelve carbon atoms (C₂₋₁₂) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted, and includesradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations. Examples include, but are not limited to,ethylenylene or vinylene, allyl, and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂₋₈) with at least one she ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl, propynyl, and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃₋₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, e.g., as a bicyclo [4,5], [5,5],[5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10 ringatoms can be arranged as a bicyclo [5,6] or [6,6] system, or as bridgedsystems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆₋₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), e.g.:a bicyclo [4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, e.g., 2-hydroxypyridinyl),imidazolyl, imidazopyridinyl, pyrimidinyl (including, e.g.,4-hydroxypyrimidinyl), pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl,tetrahydroisoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl,thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl,naphthyridinyl, and furopyridinyl. Heteroaryl groups are optionallysubstituted independently with one or more substituents describedherein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or (5-carboline.

The term “organoboron” refers to organic derivatives of boron. Examplesof organoboron compounds include esters of the following structure:

where R′ is suitably is suitably hydrocarbyl, substituted hydrocarbyl,alkyl, alkylene, carbocycle, heterocycle, aryl, heteroaryl andaryl-alkyl. Examples of R′ include ethylene (—CH₂CH₂—), neopentyl(—CH₂C(CH₃)(CH₃)CH₂—), and pinacol (—C(CH₃)(CH₃)C(CH₃)(CH₃)C—). R″ issuitably optionally substituted hydrocarbyl, substituted hydrocarbyl,alkyl, alkylene, carbocycle, heterocycle, aryl, heteroaryl andaryl-alkyl. Examples of R″ include substituted heteroaryl compoundsincluding 2-hydroxypyridine and 2-aminopyrimidine. In some particularaspects, R′ is pinacol and R″ is 2-aminopyrimidine. Other examples oforganoboron compounds include triorganoboranes and hydrides, borinic andboronic acids and esters, carboranes, and boryl compounds.

The term “volumes” refers to the amount of a first liquid compound inreference to the volume of a second compound or second mixture ofcompounds to which it is combined. For instance, four volumes of a firstliquid added to a second compound (1 volume) correlates to a volumepercent of the first liquid of 80% calculated by: (4 volumes firstliquid)/(4 volumes first liquid+1 volume second compound)*100=80 v/v %.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The terms “compound of this disclosure,” and “compounds of the presentdisclosure” and “compounds of Formulae II, IIa, III and IIIa” includecompounds of Formulae II, IIa, III and IIIa and stereoisomers, geometricisomers, tautomers, solvates, metabolites, and pharmaceuticallyacceptable salts and prodrugs thereof.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis invention, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats or prevents theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, or (in) prevents or delays the onset ofone or more symptoms of the particular disease, condition, or disorderdescribed herein. In the case of cancer, the therapeutically effectiveamount of the drug may reduce the number of cancer cells; reduce thetumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. To the extent the drug may preventgrowth and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. For cancer therapy, efficacy can be measured, e.g., byassessing the time to disease progression (TTP) and/or determining theresponse rale (RR).

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

EXAMPLES

Methods

The chemical reactions described in the Examples may be readily adaptedto prepare a number of other PI3K inhibitors of the disclosure, andalternative methods for preparing the compounds of this disclosure aredeemed to be within the scope of this disclosure. For example, thesynthesis of non-exemplified compounds according to the disclosure maybe successfully performed by modifications apparent to those skilled inthe art, e.g., by utilizing other suitable reagents known in the artother than those described, and/or by making routine modifications ofreaction conditions. Alternatively, other reactions disclosed herein orknown in the art will be recognized as having applicability forpreparing other compounds of the disclosure.

In the Examples described below, unless otherwise indicated alltemperatures are set forth in degrees Celsius. Reagents were purchasedfrom commercial suppliers, such as Sigma Aldrich Chemical Company, J. T.Baker, Boron Molecular, Mallinckrodt, and were used without furtherpurification unless otherwise indicated. The reactions set forth belowwere done generally under a positive pressure of nitrogen or argon orwith a drying tube (unless otherwise stated) in anhydrous solvents, andthe reaction flasks were typically fitted with rubber septa for theintroduction of substrates and reagents via syringe. Glassware was ovendried and/or heat dried. Column chromatography was conducted on aBiotage system (Manufacturer: Dyax Corporation) having a silica gelcolumn or on a silica SEP PAK® cartridge (Waters). ¹H NMR spectra wereobtained at 400 MHz in deuterated CDCl₃, d₆-DMSO, CH3OD or d₆-acetonesolutions (reported in ppm), using chloroform as the reference standard(7.25 ppm). When peak multiplicities are reported, the followingabbreviations are used: s (singlet), d (doublet), t (triplet), m(multiplet), br (broadened), dd (doublet of doublets), dt (doublet oftriplets). Coupling constants, when given, are reported in hertz (Hz).

HPLC may be conducted by the following exemplary methods:

LCMS short method-10 min run HPLC-Agilent 1200 Mobile phase A Water with0.05% TFA Mobile phase B Acetonitrile with 0.05% TFA Column AgilentZORBAX SD-C18, 1.8 μm, 2.1 × 30 mm Column temperature 40° C. LC gradient3-95% B in 8.5 min, 95% in 2.5 min LC flowrate 400 μL/min UV wavelength220 nm and 254 nm Mass Spec-Agilent quadrupole 6140 Ionization ESIpositive Scan range 110-800 amu Waters Acquity/LCT long method-20 minrun Waters Acquity UPLC Mobile phase A Water with 0.05% TFA Mobile phaseB Acetonitrile with 0.05% TFA Column Acquity UPLC BEH C18, 1.7 μm, 2.1 ×50 mm Column temperature 40° C. LC gradient 3-98% B in 17.0 min, 98% in1.5 min LC flowrate 600 μL/min UV wavelength 254 nm Mass Spec-Waters LCTPremier XE Ionization ESI positive Scan range 110-800 amu PhenomenexOnyx Mobile phase A 0.05% Formic Acid/Water Mobile phase B 0.05% FormicAcid/Acetonitrile Column Phenomenex Onyx Monolithic C18 column, 2 × 50mm (CV = 0.157 mL) Column temperature 35° C. Flow Rate 0.785 mL/minute(5 CV/min) Injection Volume 2 μL Sample Concentration 0.5-1.0 mg/mL in50% Acetonitrile/water Signal 220 nm Bandwidth 4 nm, Reference off Storespectrum 190-400 nm Range Step 2.0 nm Threshold 1.0 mAU Peakwidth >0.01mm Slit 4 nm

HPLC analysis of 2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol(Compound 5),2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine(Compound 7), and5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine(GDC-0084) may be done as follows. Diluent: acetonitrile. Mobile phaseA: 0.05% TFA/water. Mobile phase B: 0.05% TFA/acetonitrile. Column: Ace3 C18 HL column, 3×50 mm 3.0 μm. Column temperature: 35° C. Detectorwavelength: 220 nm. Injection volume: 2 μL. Flow rate: 1 mL/min. SampleConcentration: 0.5-1.0 mg/mL in 50% acetonitrile/water. Program: 0.0 min5.0% B, 0.3 min 5.0% B, 2.0 min 60.0% B, 4.0 min 90% B, 5.0 min 90% B,5.1 min 5.0% B, 6.5 min 5.0%. Typical retention times: 5 (RT 2.61 min),7 (RT 2.20 min), GDC-0084 (RT 2.67 min).

The structure of compound 5 was verified by NMR (see FIGS. 5 and 6 ).The structure of compound 7 was verified by NMR (see FIGS. 7 and 8 ).The structure of GDC-0084 was verified by NMR (see FIGS. 11 and 12 ).

Example 1: Preparation of2,6-dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (Compound 2) asIndicated Below

To a reactor was charged 2,6-dichloro-9H-purine (compound 1) (28.50 kg,150.79 mol, 100 mol %), EtOAc (285.00 L, 10 vol), and pyridiniump-toluenesulfonate (PPTS) (568.5 g, 2.26 mol, 1.5 mol %), followed by aslow addition of 3,4 dihydro-2H-pyran (34.25 kg, 407.16 mol, 270 mol %)at 20 to 25° C. The mixture was slowly heated to 50 to 55° C. andmaintained until HPLC analysis showed compound 1 to be no more than 1.0A % (3 h). The reaction mixture was then cooled to 20° C., washed withsaturated aqueous NaHCO₃ (81 L, 2.8 vol) and brine (81.00 L, 2.8 vol),dried over Na₂SO₄ (14.30 kg), filtered, and distilled under vacuum toremove EtOAc (230 L). The crude product was filtered and charged back tothe reactor. Hexanes (142 L) was added and the mixture was agitated for15 min. The mixture was filtered, then dried under vacuum at 55° C. for8 h to afford 39.90 kg of compound 2 (97% yield, 99 A % HPLC) as a greensolid: mp 116° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 8.92 (s, 1H), 5.72 (dd,1H), 4.01 (m, 1H), 3.72 (m, 1H), 2.25 (m, 1H), 1.98 (m, 2H), 1.74 (m,1H), 1.58 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 153.2, 151.7, 150.4,146.9, 131.1, 82.2, 68.2, 30.2, 24.8, 22.5. HRMS [M+H]⁺ calcd forC₁₀H₁₀Cl₂N₄O 273.0304; found 273.0300.

Example 2: Preparation of4-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl)morpholine(compound 3) as Indicated Below

A reactor was charged with compound 2 (39.90 kg, 146.09 mol, 100 mol %)and MeOH (399 L), then cooled to 0° C. Morpholine (38.20 kg, 438.48 mol,300 mol %) was added at 0-5° C. The reaction mixture was warmed to 20 to25° C. until HPLC analysis showed 2 to be no more than 1.0 A % (24 h).The mixture was cooled to 0 to 5° C., held for 1 h, and then filtered.The cake was washed with hexanes (200 L, 5 vol) and dried under vacuumat 55° C. for 14 h to afford 44.40 kg of compound 3 (94% yield, 99 A %HPLC) as a green solid: mp 139° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 8.39 (s,1H), 5.60 (dd, 1H), 4.20 (bs, 4H), 4.01 (m, 1H), 3.71 (m, 4H), 2.17 (m,1H), 1.94 (m, 2H), 1.74 (m, 1H), 1.58 (m, 2H): ¹³C NMR (125 MHz,DMSO-d₆) δ 153.9, 153.2, 151.6, 139.0, 118.4, 83.3, 68.2, 66.5, 45.8,30.6, 24.9, 22.7. HRMS [M+H]⁺ calcd for C₁₄H₁₈ClN₅O₂ 324.1222; found324.1222.

Example 3: Preparation of2-(2-chloro-6-morpholino-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol

2-(2-chloro-6-morpholino-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol(compound 4) was prepared from4-(2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-yl) morpholine(compound 3) as indicated below.

Compound 3 (310 g, 100 mol %) and tetrahydrofuran (4.0 L, 13 vol.) wereadded to a 12 L round bottom flask and a reaction solution was formed.The solution was cooled to −10° C. and i-PrMgCl (287 mL, 2.0 M in THF)was added while maintaining the temperature at less than or equal to −5°C. A reaction mixture was formed by combining n-BuLi (421 mL, 2.5 M inhexane) with the reaction solution while maintaining the temperature atless than or equal to −5° C. The reaction mixture was maintained at lessthan or equal to −5° C. until analysis by HPLC indicated that compound 3was no more than 5 area percent by HPLC (“A %”). Acid was added to thereaction mixture that was then quenched by adding H₂O (8.0 L, 0° C.).The layers were separated, and the aqueous phase was extracted with MTBE(1.5 L). The combined organic extracts were washed with saturatedaqueous NaCl (1.5 L), then concentrated to near dryness. i-PrOH (1.5 L)was combined with the organic extracts and the admixture wasconcentrated to near dryness. The concentrate was transferred to a 2 Lround bottom flask, combined with 2-propanol (1 L), and heated at 50° C.for 3 h. The mixture was cooled to 25° C. to form a slurry bycrystallization and held at that temperature for 12 h. The slurry wasthen cooled to 0° C., held at that temperature for 3 h, Altered toisolate a filter cake, and washed with cold 2-propanol (200 mL). Thefilter cake was dried in vacuo at 50° C. for 12 h. The process yielded329 g of compound 4 (90% yield) as an off-white solid. About a 3% lossof compound 4 was noted during crystallization.

In a scale up evaluation, to compound 3 (40.80 kg, 126.01 mol, 100 mol%) in anhydrous THF (408 L, 10 vol) was added a solution of i-PrMgCl(1.0 M in THF, 82 L, 82.00 mol, 65 mol %) at −15° C. under N₂, followedby the addition of n-BuLi (2.5 M in hexane, 55 L, 137.50 mol, 109 mol %)between −10° C. and −5° C. After 30 min at this temperature acetone(16.20 kg, 278.92 mol, 221 mol %) was added slowly to the solution. Thereaction mixture was stirred 2 h at −10±5° C. until HPLC analysis showedcompound 3 to be no more than 1.0 A % (2 h). The mixture was thentransferred to a reactor containing water (40.80 kg, 1 vol) at 0 to 5°C., the mixture was agitated for 5 min, and the layers were separated.The aqueous phase was extracted with MTBE (136 L, 3.3 vol). The combinedorganic extracts were washed with brine (119 L, 2.9 vol), dried overNa₂SO₄ (34 kg), filtered, and evaporated to provide the crude product.The yellow residue was suspended in 2-propanol (204 L, 5 vol) and theresulting slurry was heated to 50° C. for 60 min. The slurry was cooledto 25° C. over 2 h and then cooled to 0 to 5° C. over 1 h. The productwas collected by filtration, washed with cold 2-propanol (40.8 L, 1vol), and dried under vacuum at 55° C. for 12 h to afford 42.50 kg ofcompound 4 (91% yield) as an off-white powder: mp 181° C.; ¹H NMR (500MHz, DMSO-d₆) δ 6.27 (dd, 1H), 5.81 (s, 1H), 4.15 (vbs, 4H), 4.05 (m,1H), 3.69 (m, 4H), 3.55 (m, 1H), 1.97 (m, 1H), 1.70 (m, 1H), 1.60 (s,3H), 1.56 (m, 3H), 1.53 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 155.2,154.0, 153.7, 152.3, 117.1, 84.3, 70.0, 68.5, 66.5, 45.8, 30.5, 29.7,28.0, 25.0, 23.5. HRMS [M+H]⁺ calcd for C₁₇H₂₄ClN₅O₃ 382.1640; found382.1644.

Example 4: Preparation of2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol

2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol (compound 5) wasprepared from2-(2-chloro-6-morpholino-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-8-yl)propan-2-ol(compound 4) as indicated below.

Compound 4 (327 g, 100 mol %), p-toluenesulfonic acid monohydrate (4.88g, 3 mol %), and methanol (2.6 L, 8 vol) were combined in a 5 L roundbottom flask to form a solution. The solution was heated to 50° C., andthen held at that temperature for 3 h whereupon the compound 4 contentwas no more than 1 A % as measured by HPLC. The solution was cooled toambient temperature, concentrated to dryness and combined with water(1.0 L). A solid product was isolated by filtration and washed withwater (2×500 mL) and then with heptane (2×500 mL), and then dried invacuo at 50° C. for 12 h. The process yielded 246 g compound 5 as a paleyellow fluffy solid.

Example 5: Preparation of2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine

2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine(Compound 7) was prepared from2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol (Compound 5) asindicated below.

Compound 5 (240 g, 100 mol %), DMF (806 mL, 3.6 vol), cesium carbonate(303 g, 115 mol %), and 1,2-dibromoethane (139 mL, 200 mol %) werecombined in a 5 L round bottom flask. The resulting heterogeneousmixture was heated to 90° C. for 3 hours whereupon HPLC analysisindicated that compound 5 was 30 A % and compound 7 was 49 A %.Additional 1,2-Dibromoethane (69.4 mL, 100 mol %) was charged andreacted for 3 hours whereupon HPLC analysis indicated that compound 5was 28 A % and compound 7 was 54 A %. Additional cesium carbonate (355g, 135 mol %) and 1,2-dibromoethane (34.7 mL, 50 mol %) were thencharged and reacted for 3 hours whereupon HPLC analysis indicated thatcompound 5 was 2 A % and compound 7 was 87 A %. The mixture was cooledto ambient temperature, water (2.2 L) was added, and the admixture wasstirred for 12 h. Attempts to crystallize product after addition ofwater to quench the reaction was not successful as a gummy solid wasproduced EtOAc (1.3 L) was added, the layers were separated, and theorganic phase was washed with saturated aqueous NaCl (3×1.2 L). Theorganic phase was concentrated to minimum stir volume to produce asticky solid. Isopropanol (300 mL) was added to the solid, the admixturewas heated to 65° C. and held for 2 hours at temperature, and thenadmixture was cooled to ambient temperature. The resulting solids werefiltered and dried in vacuo at 50° C. for 12 h. The process yielded 147g compound 7 as a yellow crystalline solid.

Example 6: Preparation of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine(GDC-0084)

5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine(GDC-0084) was prepared from2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine(Compound 7) as indicated below.

In a Suzuki cross-coupling reaction, compound 7 (138 g, 100 mol %),5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine(“pinocolboronate”) (113 g, 120 mol %), dioxane (1.7 L), and potassiumphosphate (aq. 3.0 M, 284 mL, 200 mol %) were combined in a 3 L roundbottom flask. The structure of pinocolboronate was verified by NMR (seeFIGS. 9 and 10 ). The contents were sparged with N₂ for 30 min,PdCl₂dppf.CH₂Cl₂ (6.96 g, 2 mol %) was then charged, and the admixturewas sparged with N₂ for 10 min. The mixture was healed to 80° C. andthen held at that temperature for 2 h, after which analysis by HPLCshowed complete consumption of compound 7. The admixture was transferredto a 12 L round bottom flask, water was added (6.8 L), the mixture wascooled to 5° C., filtered, and the filter cake was washed with water(2×500 mL). The filter cake was dried in vacuo at 50° C. for 12 h. Theresulting crude GDC-0084 was transferred to a 5 L round bottom flask andcombined with THF (3.2 L), HOAc (1.6 L), and water (480 mL). Theadmixture was heated to 50° C. and Si—(CH₂)₃SH (“Si-Thiol”) (80 g) wasadded to the resulting solution. The admixture was held 3 h at 50° C.,cooled to ambient temperature and held for 12 h. The mixture wasfiltered through a pad of Celite/silica gel and the filtrate wasconcentrated to dryness. The resulting pale brown solid was transferredto a 5 L round bottom flask and combined with THF (3.2 L), HOAc (1.6 L),and water (480 mL). The admixture was heated to 50° C. and THF (100 mL)was added to obtain a clear solution. In order to improve the color ofthe solution, Si-Thiourea (80 g) was added, the mixture was held 2 h at50° C. and then filtered while hot through a 2 mm Teflon filter. Theresulting pale yellow solution was distilled to dryness, slurried withn-butanol (3 L), filtered, and washed with heptane (2×1 L). The washedfiltrate was dried in vacuo at 70° C. for 24 h. The process gave 149 gGDC-0084 (91% yield over 2 steps) as a yellow crystalline solid. Purityby HPLC was 98.9 A % with the major impurities identified as Impurity-1and Impurity-2 illustrated below, and a residual Pd level of 12 ppm.

The overall yield of GDC-0084 by the series of reaction steps ofExamples 1 to 4 was 44% with Impurity-1 present at 0.52 A % (HPLC) andImpurity-2 present at 0.18 A % (HPLC).

Example 7: Annulation of Aminoalcohol Compound 5 to Form FusedMorpholine Compound 7

The effect of solvent, base and reaction temperature for the alkylationof compounds 5 with 1,2-dibromoethane to form compound 7 was evaluated.Table 1 below lists the combination of process parameters and reagentsand associated reaction conversion for Experiments 1 to 10 whereExperiments 1 to 8 were performed on a 0.3 g scale, Experiment 9 wasdone on a 60 g scale, and Experiment 10 was done on a 100 g scale.Experiments 1 to 8 were conducted by combining the reagents (1.0 mmol ofcompound 5) with 1.5 mL solvent in vials with stir bar mixing at ambienttemperature followed by elevation to reaction temperature. The reactionsfor experiments 9 and 10 were done by mixing the reagents withmechanical mixing at ambient temperature followed by elevation toreaction temperature. For Experiment 9, 1,2-dibromoethane was addeddrop-wise over 1 hour to the heated reaction mixture and an exotherm wasobserved. For Experiment 10, compound 5 was added in two portions to theheated reaction mixture with 1 hour between additions. The totalreaction time for each experiment was 12 hours. The results are reportedin Table 5 below wherein “Exp” refers to experiment number, “DBU” refersto diazabicycloundecene, “Temp” refers to the reaction temperature, “A %sub” refers to the area percent (HPLC) of substrate2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol, “A % prod” refers tothe area percent (HPLC) of2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine,“A % Imp. 3” refers to the area percent (HPLC) of Impurity 3 (depictedbelow—area percent by HPLC after 12 hour reaction time) and “A % Imp. 4”refers to the area percent (HPLC) of Impurity 4 (depicted below—areapercent by HPLC).

TABLE 1 A % A % A % A % Exp Base Solvent Temp Sub prod Imp. 3 Imp. 4 1Cs₂CO₃ DMF  90° C. 1 86 13 0 2 K₂CO₃ DMF  90° C. 1 87 12 0 3 Cs₂CO₃ MIBK 90° C. 4 82 13 1 4 K₂CO₃ MIBK  90° C. 33 56 7 4 5 Cs₂CO₃ THF  90° C. 579 15 1 6 K₂CO₃ THF  90° C. 46 36 7 11 7 Cs₂CO₃ CH₃CN  90° C. 3 84 14 08 K₂CO₃ CH₃CN  90° C. 4 81 11 4 9 K₂CO₃ DMF 100° C. 0 85 11 0 10 K₂CO₃DMF 110° C. 0 85 11 0

Cesium carbonate and potassium carbonate were found to functionsimilarly in terms of conversion, which the latter afforded a slightlylower amount of Impurity 3. The ratio of the product to Impurity 3 wasrelatively insensitive to base, solvent and temperature. The reactionwas found to be exothermic at larger scale, and dose-control additionswere investigated to mitigate possible safety risk. Slow addition of1,2-dibromoethane to a mixture of compound 5 and K₂CO₃ (experiment 9)did not adversely affect the conversion, but did lead to the formationof impurity 5 (below). Portion-wise addition of compound 5 to a mixtureof 1,2-dibromoethane and K₂CO₃ (experiment 10) suppressed the formationof impurity 5 to less than 1 area % (by HPLC) without negativelyimpacting conversion.

The exothermic annulation reaction was further examined by reactioncalorimetry. In a first experiment, an Advanced Reactive SystemScreening Tool (ARSST™) found a reaction exotherm at 45° C. and anexothermic decomposition temperature (T_(D24)—the temperature at whichtime-to-maximum-rate is 24 hours) of 185° C. ARSTT methodology is knownin the art and is available from Fauske & Associates, Inc. See, forinstance, James P. Burlebach, “Advanced Reactive System Screening Tool(ARSST)”, North American Thermal Analysis Society, 28^(th) AnnualConference, Orlando, Oct. 4-6, 2000. In a second calorimetry experiment,integration of the reaction heat flow curve in an isothermal reactioncalorimeter at 90° C. showed an adiabatic temperature rise of 76.5° C.,an exotherm that was dose-controlled by portion-wise addition ofcompound 5. Taken together, the calorimetry data indicates that theexotherm may be effectively controlled by employing dose-controlledaddition.

Example 8: Study of Suzuki Coupling Process Parameters

Various process parameters were investigated for the Suzuki crosscoupling reaction of compound 7 with pinocolboronate to yield GDC-0084.The reactions for each experiment were done by mixing the reagentsindicated in Table 2 at ambient temperature and then heating withmixing. The reactions were done in vials by mixing the reagents (0.9mmol of compound 7) in the solvent (3.9 mL) at a solvent ratio of 10:1at ambient temperature, evacuating and backfilling with nitrogen,sealing the vials, and then heating the sealed vials. Each ofexperiments 1, 2 and 4 to 7 were reacted for 2 hours at 80° C. Theexperiment 3 reaction was run for 10 hours. The results are reported inTable 2 below wherein “Exp” refers to experiment number, “Cat %” refersto mole percent catalyst, and “Ratio” refers to the molar ratio ofproduct to substrate.

TABLE 2 Ratio Exp Cat % Catalyst Solvent Product Substrate 1 5 PdCl₂dppf· CH₂Cl₂ Dioxane, H₂O 99  1 2 2 PdCl₂dppf · CH₂Cl₂ Dioxane, H₂O 99  1 31 PdCl₂dppf · CH₂Cl₂ Dioxane, H₂O  88¹ 12 4 2 PdCl₂dppf · CH₂Cl₂ THF,H₂O 98  2 5 2 PdCl₂dppf · CH₂Cl₂ MeTHF, H₂O 91  9 6 2 PdCl₂dppf · CH₂Cl₂CH₃CN, H₂O 94  6 7 2 PdCl₂dppf · CH₂Cl₂ IPA, H₂O 93  7 ¹After 10 hoursthe ratio of product to substrate for Experiment 9 was 99:1.

In terms of catalyst loading, conversion was found to be slow at 1 mol%. The conversion was found to be high in most solvents.

Example 9: Solubility Tests

The solubility of GDC-0084 in various mono- and ternary-solvent systemswas measured at 50° C., wherein each ternary solvent mixture had asolvent ratio of 67:24:9 on a volume basis. The solubility results areindicated in Table 3 below in wt % (mg/g).

TABLE 3 Solvent Solubility Solvent Solubility DMF 0.9 wt. % DMF/HOAc/H₂O0.3 wt. % THF 0.7 wt. % THF/HOAc/H₂O 3.7 wt. % MeTHF 0.3 wt. %MeTHF/HOAc/H₂O 2.7 wt. % MeOH 0.2 wt. % MeOH/HOAc/H₂O 0.4 wt. % EtOH 0.1wt. % EtOH/HOAc/H₂O   1 wt. % n-PrOH 0.1 wt. % n-PrOH/HOAc/H₂O 1.8 wt. %i-BuOH 0.1 wt. % i-BuOHHOAcH₂O 2.1 wt. % n-BuOH 0.1 wt. %n-BuOH/HOAc/H₂O 2.4 wt. % PhMe 0.1 wt. % Toluene/HOAc/H₂O 3.8 wt. % HOAc2.8 wt. % HOAc/HOAc/H₂O 2.1 wt. %

The results indicate that toluene/HOAc/water (67:24:9) was the bestsolvent system for GDC-0084 solubility. A ratio of 69:30:1 was selectedfor scale-up evaluations.

Example 10: Study of Pd Removal

The crude GDC-0084 prepared according to example 8 was found to containelevated levels of residual palladium. In this example, removal ofpalladium from crude GDC-0084 was examined. In a series of experiments,5 g solutions of GDC-0084 in THF/HOAc (2:1 ratio) comprising 2400 ppm Pdwere exposed to a variety of metal scavengers (at 20 wt % loading) at55° C. for 14 h, followed by filtration and concentration of thefiltrate, and analysis of the resulting purified GDC-0084 for palladiumcontent. The scavengers included: 0.3-0.8 mm porous carbon beads havinga 1200 m²/g surface area (“Quadrapure C”); 100 mesh activated carbon(Darco G-60); greater than 45 μm activated carbon (“Darco KB-G”);(“SiTAACoNa”); Si—(CH₂)₃NHC(═S)NHCH₃ (“Si-Thiourea”); Si-Thiol; andpowdered synthetic magnesium-silica gel (“Si-Thiol/Florisil”). Si-Thioland Si-Thiourea are proprietary solid-supported resins available fromSilicycle. The results are presented in Table 4 below where Si-Thioureaand Si-Thiol were the most efficient scavengers.

TABLE 4 Scavenger ppm Pd Scavenger Ppm Pd None (control) 2400 SiTAAcONa900 Quadrapure C 2200 Si-Thiourea  16 Darco G-60 1600 Si-Thiol  6 DarcoKGB  900 Si-Thiol/Florisil  7

Example 11: Preparation of Purified GDC-0084

Purified GDC-0084 was prepared from compound 5 in a three step processas depicted below:

In the first step, compound 7 was prepared from compound 5. A 100 Lreactor was charged with DMF (20.0 L, 3.43 vol), 1,2-dibromoethane (7.36kg, 39.2 mol, 200 mol %), potassium carbonate (6.76 kg, 48.9 mol, 250mol %), and a first portion of compound 5 (3.01 kg, 10.1 mol, 52 mol %)(¹H NMR (500 MHz, DMSO-d6): δ 13.02 (s, 1H), 5.55 (s, 1H), 4.44-3.91 (m,4H), 3.83-3.57 (m, 4H), 1.52 (s, 6H). ¹³C NMR (125 MHz, DMSO-d6): δ157.7, 153.4, 153.0, 151.7, 117.2, 68.4, 66.0, 45.3, 29.3). Theadmixture was heated to 103° C., held at that temperature for 2 h, andthen cooled to 50° C. HPLC IPC showed compound 5 to be 0.05 A %, andImpurity 4 to be 3.9 A %. The second portion of compound 5 (2.82 kg,9.47 mol, 48 mol %) was charged to the reactor. The admixture was heatedto 85° C., held at that temperature for 16 h, then cooled to 53° C. HPLCIPC showed compound 5 to be 0.05 A % and Impurity 4 to be 0.05 A %. Thereaction was cooled to 21° C. and ethyl acetate (19.5 L) and purifiedwater (40.0 L) were added. The aqueous layer was removed and additionalethyl acetate (19.5 L) and purified water (20.0 L) were added to thereactor. The second aqueous phase was removed. GC IPC showed DMF to be0.7 wt % in the organic solution. This solution was transferred, alongwith ethyl acetate rinse (1.0 L), from the 100 L reactor to a 50 Lreactor. Distillation was carried out to minimum stir volume (9 L) andisopropanol (12 L) was charged to the reactor. Distillation was againcarried out to minimum stir volume (9 L) and 2-propanol (30 L) wasadded. GC IPC showed ethyl acetate to be 0.06 wt %. The 2-propanolsuspension (39 L) was then heated to 65° C., held at that temperaturefor 2 h, cooled to 5° C. over 1 h, and the mixture was held for 2 h atreduced temperature. HPLC IPC indicated the product concentration insupernatant to be 14 mg/g. The mixture was filtered on an Aurora filterand the cake was washed with isopropanol (16 L). After no more filtratecould be collected from the filter, the cake was dried on the filter at50±5° C. (jacket temperature) under house vacuum with a nitrogen purge.HPLC IPC showed Impurity 3 to be <1.0 A %. Drying was continued until GCIPC showed 2-propanol to be 0.91 wt %. The process gave 4.27 kg compound7 product (67% yield; 97.8 A % by HPLC) as a light yellow solid. ¹H NMR(500 MHz, DMSO-d6): δ 4.75-4.00 (m, 8H), 3.73-3.71 (m, 4H), 1.56 (s,6H). ¹³C NMR (125 MHz, DMSO-d6): δ 152.9, 151.7, 151.6, 151.5, 117.3,73.6, 66.0, 57.6, 45.3, 41.6, 27.2.

In the second step, crude GDC-0084 was prepared from compound 7. A glasscarboy was charged with K₃PO₄.H₂O (5.84 kg, 24.8 mol, 200 mol %) inpurified water (8.45 L). The contents were stirred until homogeneous,and then sparged with N₂ for ≥1 h. A 100 L reactor was charged with theaqueous K₃PO₄ solution, compound 7 from the first step (4.10 kg, 12.7mol, 100 mol %), pinocolboronate (3.36 kg, 15.2 mol, 120 mol %) (¹H NMR(500 MHz, DMSO-d6): δ 8.38 (s, 2H), 7.02 (s, 2H), 1.27 (s, 12H). 13C NMR(125 MHz, DMSO-d6): δ 164.6, 163.9, 107.8, 83.4, 24.6), and THF (55.8L). The contents were sparged with N₂ for 45 min, and thenPdCl₂dppf.CH₂C₁₋₂ catalyst (0.207 kg, 0.25 mol, 2 mol %) was charged.The admixture was sparged with N₂ for 10 min, heated to ≥61° C., andheld at that temperature for 4 h. HPLC IPC showed compound 7 to be 0.027mg/mL. Purified water (45.8 L) was added and the reaction mixture wascooled to 7° C. and held at that temperature for 1 h. The reactionmixture was filtered on an Aurora filter and the filter cake was washedwith purified water (4×30.0 L). After no more filtrate could becollected from the filter, the filter cake was dried on the filter at70° C. (jacket temperature) under house vacuum with a N₂ purge. Dryingwas continued for 8 h. HPLC IPC impurity 2 to be 0.4 A %. The contentsof the Aurora filter (crude GDC-0084) were transferred to a 100 Lreactor and combined with Si-Thiol (2.22 kg), Si-Thiourea (2.22 kg),acetic acid (17.5 L), toluene (7.5 L), and purified water (0.25 L) toform an admixture. The admixture was heated to 90° C. and held at thattemperature for 3 h. The admixture was transferred, along with aceticacid (7.5 L)/toluene (3.5 L) rinses (2×), to a Nutsche filter, and thehot filtrate (about 70° C.) was passed through an in-line filter (polishfiltration) directly into a 50 L reactor. Metal analysis IPC showedresidual Pd to be ≤3 ppm. Distillation was earned out to minimum stirvolume (10 L), and then the contents were heated to 70° C. 2-Propanol(40.0 L) was charged to the reactor through an in-line filter (polishfiltration) and the resulting suspension was heated to 70° C., held atthat temperature for 1 h, cooled to 18° C. over 4 h, and held for 9 h atreduced temperature. HPLC IPC indicated the GDC-0084 concentration insupernatant to be 1.5 mg/g. The mixture was filtered through a filterdryer and the filter cake was washed with isopropanol (48 L). After nomore filtrate could be collected from the filter, the filter cake wasdried in the filter dryer at 70° C. (jacket temperature) under housevacuum with a N₂ purge. GC IPC showed isopropanol to be 0.52 wt % andacetic acid to be 0.44 wt %. The process gave 3.87 kg GDC-0084 (80%yield in step 2; 99.4 A % by HPLC) as a light yellow solid. ¹H NMR (500MHz, DMSO-d6): δ 9.09 (s, 2H), 7.03 (s, 2H), 4.32-4.17 (m, 4H),4.17-4.04 (m, 4H), 3.84-3.65 (m, 4H), 1.58 (s, 6H). 13C NMR (125 MHz,DMSO-d6): δ 163.8, 157.6, 154.2, 152.5, 151.3, 151.0, 120.3, 117.3,73.7, 66.2, 57.8, 45.2, 41.5, 27.3.

The overall synthesis for the combination of steps 1 and 2 gave 3.87 kg(99.6% purity) of GDC-0084 at a yield of 55%. Measured impuritiesincluded Impurity 2 (0.08 A %) and Impurity 6 (0.24 A %) (depictedbelow); and the total unspecified impurities were <0.05 A %. The finalsolvent content was 1 wt. % including 0.57 wt % (i-PrOH) and 0.43 wt %HOAc, the final water content was 0.09 wt %, and the final residual Pdlevel was <3 ppm.

Example 12: Preparation of2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purine

A process for the conversion of compound 5 to compound 7 was evaluatedusing a phase transfer catalyst. More particularly, preparation ofcompound 7 by condensation of compound 5 with 1,2-dibromoethane intoluene/alkaline water solvent systems in the presence of the phasetransfer catalyst Aliquat 336 (a quaternary ammonium salt comprising amixture of C₈ and lesser amounts of C₁₀ chains; 1-octanaminium,N-methyl-N,N-dioctyl chloride) was evaluated and about a 50% conversionto compound 7 was achieved.

Solvent screens were then done for the preparation of compound 7 bycondensation of compound 5 with 1,2-dibromoethane in each ofchlorobenzene, THF, Me-THF, DCM and DCE. No conversion was achieved inDCE, partial conversion was achieved in chlorobenzene, and theconversion was less than in toluene/alkaline water for the remainder ofthe solvents.

Preparation of compound 7 by condensation of compound 5 with1,2-dibromoethane in an alkaline water solvent in the absence of anorganic solvent and in the presence of the phase transfer catalysttetrabutylammonium bromide (TBAB) was evaluated. It was discovered thatthe condensation reaction went to completion in the aqueous solvent inthe absence of an organic co-solvent. Base screening experiments weredone with the bases KOH, NaOH, K₂CO₃ and NaHCO₃ and it was found thateach base showed similar reactivity and provided for complete conversionwith a similar purity profile. KOH was selected for further evaluationdue to the highest aqueous solubility.

Reagent stoichiometry evaluations were done as summarized in Table 5below for the conversion of one equivalent of compound 5 to compound 7by condensation with 1,2-dibromoethane in an alkaline water solvent inthe presence of TBAB phase transfer catalyst. In the reactions, 0.34mmol of compound 5, KOH, TBAB and 1,2-dibromoethane were combined with10 mL solvent at ambient temperature in vials and the reaction mixturewas heated with vigorous stirring to 90° C. and held for 17 hours.Conversion was measured by HPLC. In Table 5, “Exp” refers to experiment,“1,2-DBE” refers to 1,2-dibromoethane, “equiv” refers to equivalents,and “Conv. %” refers to % conversion of2-(2-chloro-6-morpholino-9H-purin-8-yl)propan-2-ol.

TABLE 5 Exp 1,2-DBE mol % KOH mol % TBAB mol % Conv. % 1 200 200 15  522 200 400 30  45 3 400 200 30  68 4 400 400 15  87 5 400 400 30 100 6300 300 30 100

The data show that complete conversion was achieved with equimolaramounts of 1,2-dibromoethane and KOH in combination with a catalyticamount of TBAB (0.3 equiv.). Optimization of the reagent stoichiometryhelped to drive the reaction to completion. One set of optimizedconditions was determined to be equivalent amounts of 1,2-dibromoethaneand KOH (300 mol % each) and a catalytic amount of TBAB (30 mol %) inwater at 90° C. for 17 hours. It is believed that the use of excess baseand 1,2-dibromoethane minimizes the competitive generation of vinylbromide.

Example 13: Reaction Temperature Studies

Reaction temperature evaluations were done as summarized in Table 6below for the conversion of compound 5 (100 mol %) to compound 7 bycondensation with 1,2-dibromoethane (400 mol %) in an alkaline watersolvent (400 mol % KOH) in the presence of TBAB phase transfer catalyst(30 mol %) and at a 22 hour reaction time. The results are also reportedin Table 6 below, where “Exp” refers to experiment, and “A %” refers toarea percent2-chloro-6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purineas analyzed by HPLC.

TABLE 6 Exp. Temperature A % 1 90.0 80.9 2 80.0 89.9 3 70.0 91.2 4 60.092.9 5 50.0 96.0 — — —

The results indicate that a reduction of the reaction temperature offrom 90° C. to 50° C. resulted in an improved impurity profile.

Example 14: Crystallization Solvent Study

The phase transfer catalyst reactions of Examples 12 and 13 resulted inthe compound 7 product separating from the aqueous layer as an oilcontaining significant concentrations of 1,2-dibromoethane. In thosereactions, 2-propanol was added to facilitate product precipitation(crystallization). Seeding with 1% product seed crystals slightlyimproved crystallization but some oiling out of product was observed. Itwas discovered that replacement of i-propanol with ethanol allowed foran essentially clean isolation of the product as a solid from theproduct mixture, wherein a water to ethanol ratio of 1.3:1 provided thecleanest separation.

Example 15: Reaction Conditions Study

Reaction conditions found in Examples 12 to 14 that provided for thehigh relative conversions were evaluated for the preparation of compound7 by the reaction of compound 5 (480 g), 1,2-dibromoethane (300 mol %),KOH (300 mol %), TBAB (30 mol %) and H₂O (5 vol), where the equivalentamounts of reactant are based on the equivalents of starting material.The reaction was run at 50° C. for 12 hours, after which time EtOH (5.5vol) was added to crystallize the product. Compound 7 was formed in 64%yield at 99.4 A % purity by HPLC.

Example 16: Pd Catalyst Study

Various catalysts comprising palladium were evaluated for thepreparation of GDC-0084 from compound 7. For each experimental reaction,3 mmol of compound 7 was admixed with THF (8 mL), water (1.2 mL), K₃PO₄(6 mmol-200 mol %) and pinacolboronate (4.0 mmol-130 mol %). Thereaction was run at 65° C. for 5 hours. For product crystallization,water (7.1 mL) was added to the reaction product mixture at 50° C.,cured for 1 hour at temperature, and cooled to 20° C. The reactionproduct was isolated by filtration and washed 3× with 1 vol. water perwash. The results are reported in Table 7 where “Exp” refers toexperiment number; “Cat. Eq.” refers to equivalents of catalyst; “AddLig.” Refers to additional ligand in mol %; “Lig. Eq.” refers toequivalents of ligand; “Base Eq.” refers to equivalents of base; and“Conv. %” refers to percent conversion of compound 7 as determined byHPLC.

TABLE 7 Cat. Add Lig. Lig. Base Conv. Exp Catalyst Mol % Mol % Mol % mol% % 1 PdCl₂dppl · CH₂Cl₂ 1 — — 200  84% 2 Pd(amphos)Cl₂ 0.5 — — 200   0%3 PdCl₂(t-Bu2PhP)₂ 0.5 — — 200   0% 4 PdXPhos 0.5 XPhos/1 1 200 100% 5PdXPhos 0.5 XPhos/0.5 0.5 200 100% 6 PdXPhos 0.5 — — 200 100% 7 PdXPhos0.5 XPhos/0.6 0.6 200  32%

PdXPhos was the most active catalyst, providing for complete conversion,and the catalytic activity was preserved at a concentration of 0.5 mol %(0.5 mol %) even in the absence of added ligand (experiment 6).Reduction of PdXPhos catalyst loading to 0.3 mol % (0.3 mol %) reducedconversion. The Pd(amphos)C₁₂ (Experiment 2) and PdCl₂(t-Bu₂PhP)₂catalysts were inactive at the evaluated concentrations.

Example 17: Catalyst Optimization Study

The reaction conditions found in Example 16 to provide for the highestconversion were evaluated for the preparation of GDC-0084 from areaction mixture comprising compound 7 (78 g) starting material,pinocolboronate (120 mol %), THF (8 vol), H₂O (1.2 vol), PdXphos (0.5mol %), and K₃PO₄ (200 mol %). The reaction was run at 65° C. for 4hours. The reaction mixture was purified by the Si-thiol purificationmethod of Example 6, but using only 10 g Si-thiol. The Si-Thioureapurification step of Example 6 was not done in this Example. GDC-0084was formed in 94% yield at 99.3 A % purity by HPLC, wherein the residualPd was 815 ppm.

As compared to Example 6, this example replaced the PdCl₂dppf.CH₂Cl₂catalyst with the more reactive PdXPhos catalyst, reduced palladiumloading from 2 mol % (2 mol %) to 0.5 mol % (0.5 mol %), eliminated theSi-Thiourea scavenger, and reduced overall scavenger loading by about90%, and reduced total solvent by 71%, while providing for comparableyield and purity.

Example 18: Purification by Crystallization

Purification of crude GDC-0084 by crystallization may suitably be doneby crystallization from an acetic acid-water solvent. However, GDC-0084may react with acetic acid to form acetamide impurity 6.

In a first evaluation, the formation of impurity 6 in solution with ofGDC-0084 (2.6 mmol), acetic acid (3.6 mL), toluene (1.6 mL) and a traceamount of water (0.01 mL) at 90° C. over time was evaluated. The resultsare reported in Table 8 below where “% Acet.” refers to percentacetamide.

TABLE 8 Time (h) % Acet. Time (h) % Acet. 0 0    9 0.9 1  0.25 23  1.252 0.4 26  1.25 3 0.5 29 1.3 6 0.8 — —

In a second evaluation, toluene was removed and the formation ofimpurity 6 of GDC-0084 in solution with 11 volumes of acetic acid andwater at 70° C. over time was evaluated. Four ratios of acetic acid towater were evaluated including 98:2, 9:1, 4:1 and 1:1. The results aredepicted in FIG. 1 and indicate that as the amount of water increases,the amount of acetamide (impurity 6) formed after 6 hours was reduced6-fold from 0.6 area % (by HPLC) to 0.1 area %. The results furtherindicate that the acetamide impurity was less than 0.15 A % for ratiosof acetic acid to water of less than 4:1 at 70° C.

Further development indicated that GDC-0084 fully dissolved in 10 vol ofacetic acid:water (3:1) at 90° C. and crystallized out at 60° C. The 30°C. temperature width of the metastable zone was deemed to be sufficientto perform polish filtration at 90° C. Based on a Pd loading reductionof from 2 to 0.5 mol %, it was found that treatment with only 10 wt % ofSi-Thiol was sufficient to reduce residual Pd to below 10 ppm.

Under conditions derived in Examples 12 to 18 for preparing GDC-0084from compound 5 including (i) annulation of compound 5 with1,2-dibromoethane using a phase transfer catalyst in water to generatecompound 7, (ii) Suzuki cross-coupling with pinocolboronate using 0.5mol % of XPhos Pd G2 catalyst, to provide crude GDC-0084; and (iii) afinal scavenging/recrystallization from acetic acid/water providedGDC-0084 in 52% yield with 99.7 area % purity and in polymorphic form.The acetamide impurity 6 was reduced from 0.25 area % to less than 0.05area % (HPLC) by adjusting the crystallization solvent composition.

Under conditions derived in Examples 12 to 18 for preparing GDC-0084from compound 5, as compared to the preparation of GDC-0084 fromcompound 5 according to Examples 5 and 6: (i) in stage 1 (preparation ofcompound 7 from compound 5), both DMF and EtOAc were eliminated from theprocess, the total solvent volume was reduced by 54%, and allextractions and solvent exchanges were eliminated; (ii) in stage 2(reparation of crude GDC-0084 from compound 7), the total solvent volumewas reduced from 58 to 17 vol (71% reduction); and (iii) in stage 3(crude GDC-0084 purification process), toluene was eliminated and thetotal solvent volume for recrystallization was lowered from 33 to 21 vol(37% reduction). Overall, the total unit operations were reduced from 21to 7, the total solvent volume for the three stages was reduced by 64%,toluene and DMF were eliminated. As a result, the process mass intensity(PMI-see Concepcion, J., et al., “Using the Right Green Yardstick: WhyProcess Mass Intensity is Used in the Pharmaceutical Industry to DriveMore Sustainable Processes”, Org. Process Res. Dev., 2011, 912-917) wasreduced from 140 to 70, which is in the practical range for a commercialprocess (see Henderson, R. K., et al., “Lessons Learned throughMeasuring Green Chemistry Performance: The Pharmaceutical Experience”,American Chemical Society, Green Chemistry Institute, PharmaceuticalRoundtable: 2008).

Example 19: Preparation of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine

Purified GDC-0084 was prepared from compound 5 in a three step processas depicted below:

In the first step, a 100 L reactor was charged with water (45.1 kg, 4.8volumes), potassium hydroxide (5.4 kg, 96.2 mol, 304 mol %), compound 5(9.40 kg, 31.6 mol, 100 mol %), tetrabutylammonium bromide (2.92 kg,9.06 mol, 28.7 mol %) and 1,2-dibromoethane (17.7 kg, 94.2 mol, 298 mol%). The mixture was heated to 47° C. and held at that temperature for 20h. HPLC IPC showed compound 5 to be 3.1 A %. Ethanol (46.2 kg, 6.2 vol)and compound 7 seed crystals (96.6 g, 1 wt %) in ethanol (0.60 kg, 0.06volumes) were added. The contents of the reactor were held for 2 h, thencooled to 5° C. over 2 h and held for 1 h. The mixture was filteredthrough a filter dryer and washed with water (27.4 kg, 2.9 volumes).After no more filtrate could be collected from the filter, the filtercake was dried on the filter at 60° C. (jacket temperature) under housevacuum with a nitrogen purge. The process gave 6.85 kg compound 7 (6.85kg, 67% yield; 98.5 A % by HPLC) as a light yellow solid. Impuritieswere detected at low levels as follows: Compound 5 (0.43 A %);Impurity-3 (0.22 A %); Impurity-4 (0.1 A %); and Impurity-3 (0.22 A %).Melting point 147° C.; NMR (500 MHz, DMSO-d₆) δ 4.75-4.00 (m, 8H),3.73-3.71 (m, 4H), 1.56 (s, 6H); ¹³C NMR (125 MHz, DMSO-d₆) δ 152.9,151.7, 151.6, 151.5, 117.3, 73.6, 66.0, 57.6, 45.3, 41.6, 27.2. HRMS[M+H]⁺ calcd for C₁₄H₁₈ClN₅O₂ 324.1222; found 324.1225.

In the second step, a 100 L reactor was charged with water (8.00 kg, 1.2volumes), THF (39.2 kg, 6.5 volumes), potassium phosphate tribasicmonohydrate (9.51 kg, 40.4 mol, 194 mol %), compound 7 (6.75 kg, 20.85mol, 100 mol %) and pinacolboronate (5.50 kg, 24.88 mol, 119 mol %) toform an admixture. The admixture was cycled from vacuum to nitrogenthree times, and then Xphos Pd G2 (82.0 g, 0.104 mol, 0.5 mol %) wascharged. The admixture was cycled from vacuum to nitrogen three times,heated to ≥67° C., and held at that temperature for 5 h. HPLC IPCanalysis indicated complete conversion. Purified water (48.1 kg, 7.1volumes) was charged and held for 1 h at 50° C. The reaction was cooledto 20° C. over 2 h, held for more than 2 h at 20, cooled to 5° C., andheld at that temperature for 2 h. The admixture was filtered on a filterdryer and the filter cake was washed with water (37.1 kg, 5.5 volumes).After no more filtrate could be collected from the filter, the cake wasdried on the filter at 60° C. (jacket temperature) under house vacuumwith a nitrogen purge. Crude GDC-0084 was obtained as an off-white solid(7.49 kg, 94% yield; 99.4 A % by HPLC).

In the third step, a 100 L reactor was charged with water (7.75 kg, 1volume), acetic acid (60.8 kg, 7.5 volumes), crude GDC-0084 (7.70 kg,20.13 mol, 100 mol %) and silica-thiol (770 g, 10 wt %) to form andadmixture. The admixture was heated to 90° C. and then held at thattemperature for 3 h. The contents were filtered through an Aurora filterand then through a 1 μm polish filter, and the filter was rinsed withhot acetic acid (7.10 kg, 0.9 volumes). The resulting solution was thencooled to 77° C. and GDC-0084 seed crystals (82 g, 1.1 wt %) were addedas a slurry in acetic acid (69 g) and water (87 g). The contents wereheld for 1 h at 68° C. Purified water (12.0 kg, 1.6 volumes) was chargedto the slurry and the slurry was cooled to 45° C., held at 45° C. for 1h, cooled to 20° C. over 2 h, held at 20° C. for 6 h, cooled to 5° C.over 2 h and held at 5° C. for 2 h. The slurry was filtered on a filterdry er and the filter cake was washed with water (69.9 kg, 9.1 volumes).After no more filtrate could be collected from the filter, the filtercake was dried on the filter at 60° C. (jacket temperature) under housevacuum with a nitrogen purge. GDE-0084 w as obtained as an off-whitesolid (6.41 kg, 83% yield, 99.7 A %). Melting point 211° C.; ¹H NMR (500MHz, DMSO-d₆) δ 9.09 (s, 2H), 7.03 (s, 2H), 4.32-4.17 (m, 4H), 4.17-4.04(m, 4H), 3.84-3.65 (m, 4H), 1.58 (s, 6H); ¹³C-NMR (125 MHz, DMSO-d₆) δ163.8, 157.6, 154.2, 152.5, 151.3, 151.0, 120.3, 117.3, 73.7, 66.2,57.8, 45.2, 41.5, 27.3. HRMS [M+H]⁺ calcd for C₁₈H₂₂N₈O₂ 383.1938; found383.1945. The residual Pd level was below 10 ppm.

As compared to the scavenging method of Example 6, the THF waseliminated from the solvent system, the Si-Thiourea scavenging step waseliminated and the Si-thiol scavenger loading was reduced by 90%.

Example 20:5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineBlood-Brain Barrier Penetration Determination

The capability of GDC-0084 (compound IIIat) to penetrate the blood-brainbarrier (BBB) in mice was determined by evaluating the unboundbrain-to-unbound plasma concentration (Bu/Pu) ratio in female CD-1 mice.The capability of compound IIIat to penetrate the blood-brain barrier(BBB) in rats was determined by evaluating the concentration of compoundIIIat in the cerebrospinal fluid of male Sprague-Dawley rats. Theresults are presented below in Table 9.

For the mouse study, [Brain]/[Plasma] ratios were determined after anoral dose of 25 mg/kg of compound IIIat as a MCT suspension to femaleCD-1 mice. MCT refers to the indicated drug dose in 0.5% methylcelluloseand 0.2% Tween 80. [Brain]_(u) and [Plasma]_(u) refer to the unboundconcentration measured in the brain and plasma respectively. The[Brain]/[Plasma] ratios are the mean values from 3 animals per timepoint determined at both 1 hour and 6 hours after administration. Thedata show that the Bu/Pu ratio was 0.41 at both 1 and 6 h therebydemonstrating that compound IIIat is capable of substantial free brainpenetration. For the male Sprague-Dawley rat study, the concentration ofcompound IIIat in the cerebrospinal fluid (CSF) was determined and the[Bram]/[Plasma] ratio was evaluated after administration of an oral doseof 15 mg/kg of compound IIIat as a MCT suspension. [Brain]/[Plasma] anddetermined for 1 animal at each of 0.25 and 2 h and 3 at 8 hours and thedata was reported are the range across the three timepoints (average ofthe 3 animals at 8 h).

The extent of protein binding was determined in vitro, in mouse plasma(Bioreclamation, Inc., Hicksville, N.Y.) by equilibrium dialysis using aHTDialysis 96-well block (HTDialysis® LLC; Gales Ferry, Conn.). Thecompound was added to pooled plasma from multiple animals (n≥3) at atotal concentration of 10 μM. Plasma samples were equilibrated withphosphate-buffered saline (pH 7.4) at 37° C. in 90% humidity and 5% CO₂for 4 hours. Following dialysis, concentration of compounds in plasmaand buffer were measured by LC-MS/MS. The percent unbound in plasma wasdetermined by dividing the concentration measured in the post-dialysisbuffer by that measured in the post-dialysis plasma and multiplying by100. Incubations were performed in triplicate and coefficient ofvariation is not greater than 30%.

The free fraction in mouse brain was determined as described by Kalvass.Briefly, brain tissue was homogenized in 3 volumes of phosphate-bufferedsaline and compound was added at a final concentration of 10 μM.Aliquots of 300 μl were dialyzed in a RED device (Thermo Scientific,Rockford, Ill.) against a volume of 500 μl buffer for 4 h at 37° C. inan incubator at 90% humidity and 5% CO₂. Following dialysis, tissues andbuffer samples were analyzed as described for the plasma protein bindingstudies.

Twelve female CD-1 mice (Charles River Laboratories, Hollister, Calif.)were given an oral (PO) dose of the indicated compound in 0.5%methylcellulose/0.2% Tween 80 (MCT). Two blood samples of approximately0.15 mL were collected from each mouse (n=3 mice per timepoint) byretro-orbital bleed or terminal cardiac puncture while the animals wereanesthetized with isoflurane. Blood samples were collected in tubescontaining K2EDTA as the anticoagulant, predose and at 0.083, 0.25, 0.5,1, 3, 6, 9, and 24 h post-dose. Samples were centrifuged within 1 h ofcollection and plasma was collected and stored at −80° C. untilanalysis. Total concentrations of the compound were determined by liquidchromatography-tandem mass spectrometry (LC-MS/MS), following plasmaprotein precipitation with acetonitrile, and injection of thesupernatant onto the column, a Varian MetaSil AQ C18 column (50×2 mm, 5μm particle size). A CTC HTS PAL autosampler (LEAP Technologies, ChapelHill, N.C.) linked to a Shimadzu SCL-10A controller with LC-10AD pumps(Shimadzu, Columbia Md.), coupled with an AB Sciex API 4000 triplequadrupole mass spectrometer (AB Sciex, Foster City, Calif.) were usedfor the LC-MS/MS assay. The aqueous mobile phase was water with 0.1%formic acid and the organic mobile phase was acetonitrile with 0.1%formic acid. The lower and upper limits of quantitation of the assaywere 0.005 μM and 10 μM, respectively. The total run time was 1.5 minand the ionization was conducted in the positive ion mode. Where brainconcentration was determined, brains were collected at 1 and 6 hpost-dose from 3 different animals at each time point, rinsed withice-cold saline, weighed and stored at −80° C. until analysis. Forcompound quantitation, mouse brains were homogenized in 3 volumes ofwater. The homogenates were extracted by protein precipitation withacetonitrile. LC-MS/MS analysis was conducted as described for theplasma. Brain homogenate concentrations were converted to brainconcentrations for the calculations of brain-to-plasma ratios.

The results are shown in Table 9 and the total brain-to-plasma ratio was1.4 for mice and 1.9-3.3 for rats. The [Brain]_(u)/[Plasma]_(u) for micewas 0.41 and the [CSF]/[Plasma]_(u) for rats was 0.73-1.0. Althoughbrain protein binding was not measured for rats, the CSF concentrationhas been established as a surrogate for unbound brain concentration. SeeLiu, X., et al., Unbound Drug Concentration in Brain Homogenate andCerebral Spinal Fluid at Steady State as a Surrogate for UnboundConcentration in Brain Interstitial Fluid, Drug Metab. Dispos. 2009, 37,787-793. The [CSF]/[plasma]_(u) concentration ratio was 0.73-1.0,indicating that compound IIIat effectively crosses the BBB in rats.

TABLE 9 Species [Brain]/[Plasma] [Brain]_(u)/[Plasma]_(u)[CSF]/[Plasma]_(u) Mouse 1.4 0.41 — Rat 1.9-3.3 — 0.73-1.0 

The effect of compound IIIat on pAKT in normal brain tissue, expressedas the ratio of phosphorylated AKT (pAKT) to total AKT (tAKT) wasevaluated. ART is critical for proliferation and antiapoptotic signalingpathways, and increased activation of AKT by phosphorylation has beenfound to be involved in a variety of neoplasia. In the evaluation,female CD-1 mice were administered a single PO dose of the indicatedcompound. Brains and plasma were collected at the indicated timepost-dose, from 3 animals at each time point. Individual brains weresplit in half for PD analysis and compound concentration measurement.The samples were stored at −70° C. and analyzed for total concentration.For PD analysis, cell extraction buffer (Invitrogen, Camarillo, Calif.)containing 10 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mMNaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1%SDS, and 0.5% deoxycholate was supplemented with phosphatase, proteaseinhibitors (Sigma, St. Louis, Mo.) and 1 mM PMSF and added to frozenbrain biopsies. Brains were homogenized with a small pestle (Konte GlassCompany, Vineland, N.J.), sonicated briefly on ice, and centrifuged at20,000 g for 20 min at 4° C. Protein concentration was determined usingBCA protein assay (Pierce, Rockford, Ill.). Proteins were separated byelectrophoresis and transferred to NuPage nitrocellulose membranes(Invitrogen, Camarillo, Calif.). Licor Odyssey Infrared detection system(Licor, Lincoln, Nebr.) was used to assess and quantify proteinexpression. PI3K pathway markers were evaluated by immunoblotting usingantibodies against pAkt^(ser473) and total Akt (Invitrogen, Camarillo,Calif. and Cell Signaling, Danvers, Mass.). Inhibition of pAkt (%) wascalculated by comparing pAkt signal with that measured in untreatedmice.

The results are shown in FIG. 2 for a 3 mg/kg or 10 mg/kg dose ofcompound IIIat administered orally where pAKT in normal mouse braintissue was measured and determined to be inhibited at 1 h post-dose. At4 hours post-dose, the 3 mg/kg dose no longer resulted in inhibition ofpAKT, in contrast to the 10 mg/kg dose. The results demonstrate thatcompound IIIat engages its target behind a fully intact BBB, thereforefreely penetrating mouse brain.

Example 21: Efficacy Evaluation of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine(GDC-0084) Against Glioblastoma

The in vivo efficacy of compound GDC-0084 (compound IIIat) versus U87 MGMerchant (MG/M) human glioblastoma xenografts was evaluated in doseescalation studies in subcutaneous tumor-bearing Taconic female NCR nudemice.

All in vivo studies were conducted in compliance with Genentech'sInstitutional Animal Care and Use Committee. PTEN-null U-87 MG/M humanglioblastoma cancer cells (an in-house derivative of U-87 MG cells fromAmerican Type Culture Collection (Manassas, Va.)) were cultured in RPMI1640 media plus 1% L-glutamine with 10% fetal bovine serum (HyClone;Waltham, Mass.). Cells in log-phase growth were harvested andresuspended in HBSS:Matrigel (BD Biosciences; Franklin Lakes, N.J.)(1:1, v:v) for injection into female NCr nude mice (Taconic Farms,Cambridge City, Ind.) aged 20 weeks. Animals received five million cellssubcutaneously in the right lateral thorax in 0.1 mL. Mice bearingestablished tumors in the range of 200-500 mm³ were separated intogroups of equally sized tumors (n=6-7/group) to receive escalating dosesof 16. The inhibitor was formulated once weekly in 0.5% methylcelluloseand 0.2% Tween-80 at concentrations needed for target doses in a volumeof 0.2 mL. All formulations were stored in a refrigerator and brought toroom temperature and mixed well by vortex before oral administration bygavage once daily for 23 days. Tumor volumes were calculated fromperpendicular length and width caliper measurements using the formula:Tumor Volume (mm³)=0.5×(Length×Width²). Changes in body weights arereported as a percentage change from the starting weight.

A mixed modeling approach was used to analyze the repeated measurementof tumor volumes from the same animals over time since this approachaddresses both repeated measurements as well as modest dropouts beforestudy end (Pinheiro et al. 2008). Log 2(tumor volume) growth traces werefitted to each dose group with restricted cubic splines for the dose andfixed time effects. Fitting was done via a linear mixed-effects model,using the R package nlme (version 3.1-97) in R version 2.13.0 (RDevelopment Core Team 2008; R Foundation for Statistical Computing;Vienna, Austria). Fitted tumor volumes were plotted in the natural scalein Prism (version 5.0b for Mac) (GraphPad Software; La Jolla, Calif.).Linear mixed-effects analysis was also employed using R to analyze therepeated measurement of body weight changes from the same animals overtime.

Mice bearing the tumor xenographs were dosed at 0 time, 2 days, 4 days,7 days, 9 days, 11 days 13 days, 16 days, 19 days and 22 days at acompound IIIat dosage rate of 0.45 mg/kg, 2.2 mg/kg, 4.5 mg/kg, 8.9mg/kg, 13.4 mg/kg or 17.9 mg/kg where compound IIIat was a suspension invehicle (0.5% methylcellulose/0.2% Tween-80). The mice control group wasadministered the vehicle in the absence of the drug once at the samedosage schedule. Changes in tumor volumes over time by dose for eachcompound are depicted in FIG. 3 as cubic spline fits generated viaLinear Mixed Effects analysis of log-transformed volumes.

Compound IIIat achieved significant and dose-dependent tumor growthinhibition. Tumor growth inhibition was first observed at a 2.2 mg/kgdose level. Higher doses led to greater tumor growth inhibition,including tumor regressions at the 17.9 mg/kg dose level. Each of thesedoses was well tolerated for the duration of the study. Compound IIIatwas found to have an anti-proliferation EC₅₀ of 740 nM in U87 cells.

Example 22: Efficacy Evaluation of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6H-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amineon pAKT

The effect of compound IIIat on the pharmacodynamics (PD) marker pAKT inthe U87 MG/M human glioblastoma xenograft model after 24 days ofcontinuous dosing at dosage rates of 0.5 mg/kg, 3 mg/kg, 10 mg/kg and 18mg/kg was evaluated. Tumors were excised from animals 1 hour and 4 hoursafter the last administered dose on day 24 and processed for analysis ofpAKT and total AKT. The results are reported in FIG. 4 as a ratio ofpAKT to total AKT wherein indicated values are the means for groups of 3animals and error bars indicate ±standard error of the mean. Levels ofpAKT^(Ser473) and total AKT were measured by electrochemiluminescenceusing Meso Scale Discovery according to manufacturer's instructions(Gaithersburg, Md.).

Compound IIIat was found to have a significant PD effect in the U87tumors. Dose and concentration dependent inhibition of pAKT was observedat both 1 hour and 4 hours post dose, indicating that tumor growthinhibition is the result of on-target inhibition.

Example 23: Assessment of Kinase Inhibition by GDC-0084

Inhibition of 229 kinases by GDC-0084 (i.e., compound IIIat) Class IPI3K Kiapp's for GDC-0084 was evaluated. The percent inhibition at 1 μMof GDC-0084 against 229 kinases is reported in Table 10 below:

TABLE 10 Kinase % inhib ACVR1B 1.1 AKT1 0.7 AKT2 6.1 AKT3 5.9 ALK 2.3Abl 2.0 Arg 14.1 Aurora_A 3.2 Aurora_B −9.1 Aurora_C −2.5 Axl 6.0 B-Raf7.6 Blk 28.8 Bmx 20.8 BrSK1 1.6 Brk 5.9 CDK1/cyclinB 3.3 CDK2/cyclinA−2.4 CDK5/p25 0.3 CDK5/p35 7.5 CDK7/cyclinH 2.4 CDK9/cyclinT1 −0.9 CHK10.6 CHK2 11.3 CK1_alpha1 1.9 CK1_epsilon1 4.8 CK1_gamma1 6.8 CK1_gamma21.4 CK1_gamma3 −0.2 CK2_alpha1 3.2 CK2_alpha2 2.3 CLK1 4.0 CLK2 5.3 CLK37.0 CSF1R 21.9 CSK 10.0 CaMKI −1.4 CaMKII_beta 0.2 CaMKI_delta 11.6CamKII_alpha 2.1 CamKII_delta −1.4 CamKIV 0.2 Cot 28.7 DAPK1 −1.2DCAMKL2 3.3 DNA-PK 17.7 DYRK1A 2.8 DYRK1B −2.7 DYRK3 −10.8 DYRK4 4.6EGFR 2.5 ERK1 8.6 ERK2 5.2 EphA1 7.8 EphA2 −0.7 EphA4 3.0 EphA5 5.9EphA8 6.9 EphB1 1.9 EphB2 6.6 EphB3 −0.1 EphB4 4.8 ErbB2 8.5 ErbB4 7.0FAK −1.9 FAK2 4.6 FGFR1 −6.8 FGFR2 2.3 FGFR3 11.5 FGFR4 5.8 Fer 8.9 Fes−10.1 Fgr 37.2 Flt1 0.6 Flt3 21.9 Flt4 4.8 Frk 8.0 Fyn 9.0 GCK 0.9 GRK2−3.6 GRK3 4.6 GRK4 −4.2 GRK5 −10.0 GRK6 2.9 GRK7 −9.5 GSK3_alpha 2.5GSK3_beta 1.0 HIPK1 3.3 HIPK2 1.6 HIPK4 2.6 Haspin 3.8 Hck 34.0 Hyl 3.3IGF1R 4.9 IKK_alpha −3.8 IKK_beta 2.3 IKK_epsilon 2.9 IRAK4 10.2 IRR11.5 InsR 6.0 Itk 9.6 JAK1 −1.6 JAK2 15.2 JAK3 8.9 JNK1_alpha1 −4.2 JNK29.8 JNK3 −2.7 KDR −3.6 KHS1 1.5 Kit 14.1 LRRK2 10.1 LTK 7.8 Lck 38.0 Lyn23.3 LynB 24.9 MAPKAPK2 1.1 MAPKAPK3 4.4 MARK1 4.4 MARK2 5.5 MARK3 4.3MARK4 1.5 MEK1 −0.1 MEK2 8.1 MELK −10.4 MLK1 28.9 MRCK_alpha −4.5 MSK110.9 MSK2 0.6 MSSK1 11.1 MST1 5.0 MST2 −2.0 MST3 −1.3 MST4 −1.5 MYLK23.0 (skMLCK) Mer 8.1 Met 6.5 Mink1 14.3 MuSK 12.7 NEK1 −6.4 NEK2 20.0NEK4 7.8 NEK6 13.1 NEK7 −0.3 NEK9 −2.6 PAK1 7.0 PAK2 3.0 PAK3 −4.5 PAK415.2 PAK6 15.3 PAK7 18.6 PASK −5.3 PDGFR_alpha 9.7 PDGFR_beta 4.8 PDK113.4 PDK1(direct) −8.8 PI3KC2a 13.2 PI3KC2b 42.6 PI3KC3_hVPS34 23.6PI4Ka 7.0 PI4Kb 2.8 PIM1 9.7 PIM2 −4.4 PKA 8.2 PKC_alpha 7.5 PKC_beta112.5 PKC_beta2 11.9 PKC_delta 1.7 PKC_epsilon 10.7 PKC_eta −9.0PKC_gamma 23.8 PKC_iota 6.8 PKC_theta 5.8 PKC_zeta 1.6 PKD1 1.6 PKD2 4.5PKD3 19.9 PKG1_alpha −3.4 PKG2 12.1 PLK1 3.3 PLK2 7.2 PLK3 −4.8 PRK1−12.7 PRKAA1 9.3 PRKAA2 5.8 PhK_gamma1 2.0 PhK_gamma2 0.0 PrKX 8.3 RAF142.3 (Y340D, Y341D) ROCK1 1.3 ROCK2 −16.1 Ret 13.4 Ron 8.0 Ros −6.7 Rse3.6 Rsk1 −4.6 Rsk2 1.4 Rsk3 4.4 Rsk4 27.4 SGK1 13.9 SGK2 2.8 SGK3 −3.5SIK2 6.8 SPHK1 −2.8 SPHK2 −2.3 SRPK1 5.5 SRPK2 −7.7 Src 29.9 Src_N1 43.6Srm −1.1 Syk 42.8 TAO1 −1.3 TBK1 4.0 TSSK1 5.5 TSSK2 −6.8 TYK2 8.4 Tie27.4 TrkA 7.3 TrkB 4.2 TrkC 11.7 YSK1 −8.8 Yes 31.3 ZAP-70 −0.9 eEF-2K5.2 p38_alpha −9.4 p38_alpha(direct) 4.3 p38_beta 6.5 p38_delta 12.1p38_gamma 11.9 p70S6K 6.0

The selectivity of GDC-0084 for Class I PI3K kinases was evaluated andthe results are reported in Table 11 below:

TABLE 11 Class I Selectivity PI3K Kinase (Ki_(app)) PI3Kα  2 nM PI3Kβ 46nM P13Kδ  3 nM PI3Kγ 10 nM mTOR 70 nM

Example 24: Stability Evaluation of5-(6,6-dimethyl-4-morpholino-8,9-dihydro-6h-[1,4]oxazino[3,4-e]purin-2-yl)pyrimidin-2-amine

The hepatocyte stability of compound IIIat was evaluated acrosspreclinical species. Certain in vivo pharmacokinetic parameters werealso evaluated. In the example, hepatic clearance was predicted fromhepatocyte incubations using the in vitro t_(1/2) method disclosed byObach, R. S., et al., The prediction of human pharmacokinetic parametersfrom preclinical and in vitro metabolism data, J. Pharmacol. Exp. Ther.1997, 283, 46-58. Male Sprague-Dawley rats, female CD-1 mice, malecynomolgus monkeys and beagle dogs were dosed intravenously with 1 mg/kgof compound IIIat prepared in 60% PEG400/10% Ethanol. Compound IIIat wasadministered orally (PO) at the indicated dose in 0.5% methylcellulosewith 0.2% Tween 80 (MCT). The results are reported in Table 12 belowwhere “Cyno” refers to cynomolgus monkeys; “Cl_(hep)” refers tohepatocyte clearance in mL/min/kg; “in vivo Cl” refers to in vivoclearance after IV administration in mL/min/kg; IV dosage was 1 mg/kg;“Vss” refers to the apparent volume of distribution at steady state inL/kg; oral dose is reported in mg/kg; “Cmax” refers to the peak serumconcentration reported in μm; “AUC” refers to the area under the curvein a plot of concentration of compound IIIat in blood plasma versus timeand is reported in μm·h; “F %” refers to percentage drugbioavailability; and “PPB %” refers to the percentage of the drug thatbinds with blood plasma protein.

TABLE 12 IV PO Species Cl_(hep) In vivo Cl Vss Dose Cmax AUC F % PPB %Mouse 30 17 1.7 25 4.6 47 75 78 Rat 3 28 3.2 5 1.1 8.3 77 71 Cyno 26 462.9 2 0.03 0.11 6 75 Dog 13 26 3.0 2 0.2 1.6 40 66

With the exception of rat, there was a good correlation betweenpredicted clearance based on hepatocyte stability and in vivo clearance.

Example 25: Human Phase I Trial

An open-label, multicenter, Phase I, dose-escalation study was doneusing a standard “3+3” design to assess the safety, tolerability, andpharmacokinetics of GDC-0084 (compound Formula IIIat). GDC-0084 is apotent, oral, selective small molecule inhibitor of class I PI3K andmTOR kinase with a mean apparent inhibition constant (Ki) forp110α/p85α, p110p/p85 α, p110δ/p85 α, and p110γ of 2.2, 41, 2.7, and 9.7nM, respectively.

GDC-0084 was administered orally once daily in continuous dosing cyclesof 28 days to a set of forty-seven patients with progressive orrecurrent high-grade gliomas (WHO Grade III-IV) who had progressedduring or after treatment with at least one priorradiotherapy-containing regimen for gliomas and/or were not candidatesfor regimens known to provide clinical benefit. GDC-0084 was provided incapsule formulations in three strengths: 1 mg, 5 mg, and 25 mg. GDC-0084capsules were stored at room temperature (59° F.-86° F. [15° C. and 30°C.]). Plasma samples for pharmacokinetic (“PK”) analysis were collectedon day 1 and day 8 or day 15 of cycle 1. Fluorodeoxyglucose positronemission tomography (“FDG-PET”) was performed at baseline andon-treatment.

The median time from primary diagnosis was 40.5 months (range: 11-190months). At study enrollment, 33 patients (70.2%) were classified withWHO Grade IV glioma and 14 patients (29.8%) were classified with WHOGrade III glioma. Of the patients, 55.3% of patients had progressivedisease, 40.4% had stable disease, one patient was not evaluable, andthe data for one patient was missing. Overall, all patients had receivedprior cancer surgery, radiotherapy, and systemic therapies. The mediannumber of prior cancer surgeries was 2.0 (range: 1 to 6), the mediannumber of prior radiotherapies was 1.0 (range: 1 to 2), and the mediannumber of prior systemic therapies was 3.0 (range: 1 to 5).

Patients received GDC-0084 daily in cycles of 28 days in length (4 weeksof daily dosing). On Day 1 of Cycle 1, GDC-0084 was administered in aclinical setting that accommodated frequent blood draws over a period ofup to 24 hours after the morning dose was administered. Patients tookGDC-0084 at the same time of day (±2 hours) when the study drug wastaken at home. Dosing times may have been adjusted to accommodate fortime shifts from the home administration schedule (e.g., for clinicvisits with PK sampling or traveling), but times were to be adjusted byno more than 4 hours at a time.

GDC-0084 was taken on an empty stomach (i.e., approximately 1 hourbefore or 2 hours after a meal) unless the patient was otherwiseinstructed, except on days when administration was under fastedconditions (e.g., with extensive PK sampling during Cycle 1, asdescribed elsewhere herein). For administration under fasted conditions,patients fasted overnight for at least 8 hours before dosing and 2 hoursafter dosing. GDC-0084 capsules were swallowed whole (not chewed) with240 mL (8 oz) of water.

Dose escalation continued in accordance with the dose-escalation rulesuntil the maximum tolerable dose (“MTD”) was exceeded, excessive pillburden (defined as 2 or more patients in a cohort who were unable totake ≥90% of doses consisting of a minimum of eight capsules) wasdeclared, or analysis of available PK data indicated that exposure wasunlikely to increase with further increases in the dose of GDC-0084.

In PK evaluations, the patients were treated with GDC-0084 in eight dosegroups on a 28-day (once daily) cycle at the following dose levels:Cohort 1 (2 mg); Cohort 2 (4 mg); Cohort 3 (8 mg); Cohort 4 (15 mg);Cohort 5 (20 mg); Cohort 6 (30 mg); Cohort 7 (45 mg); and Cohort 8 (65mg). PK evaluations were conducted following GDC-0084 administration ina fasted state on Study Days 1 and 8 (for Cohorts 7-8) or 15 (forCohorts 1-6). A single dose of GDC-0084 was administered orally on Day 1of Cycle 1, followed by frequent blood sampling, up to 72 hours forCohorts 1-6 and 24 hours for Cohorts 7-8, to determine the single-dosePK properties of GDC-0084. For Cohorts 1-6 (2-30 mg), the single dose onCycle 1, Day 1 was followed by a 7 day washout-period, after whichcontinuous once daily dosing, for 28 consecutive days, was started onDay 8. Blood samples for Cohorts 1-6 were collected on Day 15 for PKanalysis. For Cohorts 7 and 8 (45-65 mg), subjects were dosedcontinuously once daily for 28 days starting on Cycle 1, Day 1 and bloodsamples were collected on Day 8 for multiple dose PK analysis. Avalidated LC-MS/MS assay with a lower level of quantification (LLOQ) of0.00052 μM was used to quantify the concentration of GDC-0084 in plasmasamples.

Plasma concentration-time data for GDC-0084 were tabulated, anddescriptive statistics were computed and compared between cohorts. Meanplasma GDC-0084 concentration data were plotted by cohort relative tonominal time. All plasma concentration-time data collected in Cycle 1were analyzed using WinNonlin® (Version 6.4, Pharsight Corp, MountainView, Calif.) to estimate PK parameters, which included but were notlimited to AUC_(0-last) (where AUC refers to the area under theconcentration-time curve) and/or AUC_(inf), C_(max), C_(min), t_(max),half-life, CL/F, and accumulation ratio. Estimates for each PK parameterand summary statistics (mean, standard deviation, coefficient ofvariation, median, minimum, and maximum) were tabulated by dose leveland schedule. Nominal time data were used in the analysis, and thelinear up/log down trapezoidal method was used for calculating AUC.

The pharmacokinetic parameters of GDC-0084 following single and multipledoses are tabulated in Table 13 and Table 14, respectively, where SDrefers to standard deviation; % CV refers to the coefficient ofvariation; ND refers to not determined; T_(1/2) refers to terminalhalf-life; T_(max) refers to time to maximum plasma concentration;C_(max) refers to maximum observed plasma concentration; AUC_(inf)refers to area under the concentration-time curve from Time 0 toinfinity; CL/F refers to apparent oral clearance; AUC₀₋₂₄ refers torefers to area under the concentration-time curve from Time 0 to 24hours; C_(min) refers to minimum concentration; and Accumulation Ratiorefers to AUC_(0-24 hr multiple dose)/AUC_(0-24 hr single dose).

TABLE 13 PK Parameter Results after Single Dose (Cycle 1, Day 1) 2 mg 4mg 8 mg 15 mg 20 mg 30 mg 45 mg 65 mg Parameter n = 7 n = 4 n = 5 n = 6n = 4 n = 7 n = 8 n = 6 T_(1/2) (hr) Mean 16.9 21.8 18.2 18.1 14.8 22.0ND ND SD 7.38 4.41 8.94 14.4 2.96 8.66 ND ND % CV 43.6 20.2 49.2 79.620.0 39.4 ND ND T_(max) (hr) Median 2.0 3.0 3.0 2.0 2.0 2.0 3.0 2.5Range 2.0-4.0 2.0-3.0 2.0-3.0 2.0-4.0 2.0-3.0 1.0-8.0 2.0-4.0 2.0-4.0C_(max) (μM) Mean 0.0177 0.0359 0.0452 0.0912 0.159 0.174 0.234 0.255 SD0.0055 0.00468 0.00838 0.0278 0.0655 0.0483 0.0905 0.113 % CV 31.1 13.018.5 30.5 41.2 27.8 38.7 44.3 AUC_(inf) (μM*hr) Mean 0.365 0.833 0.9741.97 2.75 5.33 ND ND SD 0.190 0.122 0.568 1.60 0.932 3.59 ND ND % CV52.1 14.6 58.4 81.2 33.9 67.4 ND ND CL/F (L/hr) Mean 13.1 4.87 12.4 11.17.92 8.22 ND ND SD 21.1 0.634 10.4 5.79 2.63 5.26 ND ND % CV 161 13.084.0 52.2 33.2 64.0 ND ND AUC₀₋₂₄ (μM*hr) Mean 0.210 0.435 0.509 1.091.90 2.42 3.12 4.06 SD 0.0881 0.0221 0.176 0.459 0.654 0.945 1.10 1.75 %CV 41.9 5.08 34.6 42.2 34.5 39.1 35.3 43.1

TABLE 14 PK Parameter Results after Multiple Doses (Cycle 1, Day 15 forCohorts 1-6, Cycle 1, Day 8 for Cohorts 7 & 8) 2 mg 4 mg 8 mg 15 mg 20mg 30 mg 45 mg 65 mg Parameter n = 6 n = 4 n = 4 n = 6 n = 4 n = 6 n = 8n = 5 T_(max) (hr) Median 2.0 2.0 2.5 3.0 2.0 2.0 3.5 3.0 Range 1.0-3.02.0-3.0 2.0-3.0 2.0-4.0 2.0-3.0 2.0-3.0 3.0-4.0 1.0-3.0 C_(max) (μM)Mean 0.0331 0.593 0.0883 0.156 0.230 0.332 0.544 0.580 SD 0.0114 0.002150.0256 0.0857 0.0735 0.266 0.252 0.351 % CV 34.3 3.63 29.0 55.0 31.980.1 46.3 60.5 C_(min) (μM) Mean 0.00877 0.0207 0.0301 0.0581 0.06350.155 0.206 0.271 SD 0.00495 0.00631 0.0148 0.0651 0.0195 0.158 0.09880.145 % CV 56.4 30.4 49.2 112 30.7 102 48.0 53.6 AUC₀₋₂₄ (μM*hr)^(a)Mean 0.346 0.833 1.16 2.34 2.87 5.67 8.06 9.01 SD 0.178 0.166 0.380 1.840.499 5.79 2.76 4.62 % CV 51.4 19.9 32.8 78.6 17.4 102 34.2 51.3Accumulation Ratio^(a) Mean 1.68 1.91 1.97 2.03 1.66 1.96 2.83 2.44 SD0.328 0.327 0.342 0.781 0.650 1.41 1.19 0.775 % CV 19.5 17.1 17.3 38.439.2 71.9 42.0 31.7 ^(a)For Cohort 6 (30 mg), n = 5.

Concentration-time profiles of GDC-0084 following single and multipledoses are presented in FIG. 13 and FIG. 14 , respectively. FIG. 13 is aplot of mean±SD plasma concentration vs. time profiles of GDC-0084following a single dose (cycle 1, day 1). FIG. 14 is a plot of ±SDplasma concentration vs. time profiles of GDC-0084 following multipledoses (cycle 1, day 15 for cohorts 1 to 6 and cycle 1, day 8 for cohorts7 and 8). Following a single oral dose, GDC-0084 w as rapidly absorbedwith a median T_(max) of approximately 2 hours (range 1 to 8 hours).After reaching peak plasma concentrations, concentrations decreased withan apparent terminal phase t_(1/2) of approximately 18.73 hours (range3.41 to 47.3 hours; calculated across Cohorts 1-6 (2 to 30 mg) followinga single dose).

FIG. 15 is a GDC-0084 dose proportionality plot of dose (mg) versusC_(max) (μM) for single dose (“SD”) and multiple dose (“MD”) regimens.FIG. 16 is a GDC-0084 dose proportionality plot of dose (mg) versusAUC₂₄ (μM*hr) for single dose (“SD”) and multiple dose (“MD”) regimens.The data indicate that the accumulation ratio(AUC_(0-24 hr multiple dose)/AUC_(0-24 hr single dose)) ranged from0.577 to 4.84 with a mean value of 2.12±0.896. Both C_(max) and AUC₀₋₂₄for Cycle 1, Day 1 appeared to increase in a dose-proportional and doselinear fashion across all cohorts for both single and multiple doses.FIG. 17 is a plasma GDC-0084 mean single dose (SD) concentration versustime log scale plot (cycle 1, day 1). FIG. 18 is a plasma GDC-0084 meansingle dose (SD) concentration versus time linear scale plot (cycle 1,day 1). FIG. 19 is a plasma GDC-0084 mean single dose (SD) concentrationversus time log scale plot (cycle 1, day 8/15). FIG. 20 is a plasmaGDC-0084 mean single dose (SD) concentration versus time linear scaleplot (cycle 1, day 8/15). FIG. 21 is a log scale plot of AUC₀₋₂₄ (μM*hr)versus dose (mg) for GDC-0084 for single dose (SD) and multiple dose(MD) regimens. FIG. 22 is a log scale plot of C_(max) (μM) versus dose(mg) for GDC-0084 for single dose (SD) and multiple dose (MD) regimens.

Overall, the concentration from brain tumor tissue suggests thatGDC-0084 crosses the blood brain barrier and uniformly distributesthroughout the brain. The experimental results indicate that GDC-0084inhibited human mTOR, with a mean apparent Ki of 70 nM. GDC-0084 israpidly absorbed and demonstrates linear and dose proportional increasesin exposure, with a half-life (t_(1/2) of about 19 hours) supportive ofonce daily dosing. The MTD was determined to be 45 mg when GDC-0084 wasadministered orally once daily in cycles of 28 days. At a dose of 45 mg,steady-state concentrations were consistent with antitumor activityobserved in xenograft models. Of the patients who underwent FDG-PETimaging, 7 of 27 patients had metabolic partial response according topre-defined criteria. Of the 34 patients with exploratory MRI results,none of the MRI derived metrics (Ktrans, Cerebral blood volume, apparentdiffusion coefficient) showed any significant trend with drug plasmaexposure. GDC-0084 was rapidly absorbed with a median T_(max) ofapproximately 2 hours following a single dose. The accumulation ratiohad a mean value of 2.1±0.90, and the extent of accumulation wasconsistent with the theoretical accumulation based upon half-lifeestimates and the daily dosing interval. GDC-0084 displayed anapproximately linear and dose proportional increase in C_(max) andAUC₀₋₂₄ following single and multiple doses across all cohorts (2 mg to65 mg once daily).

Tumor response was determined by either an assessment of FDG-PET or byResponse Assessment in Neuro-Oncology (RANO).

FDG-PET assessments were used to evaluate the inhibition of glucoseuptake and will be used as a surrogate assay to address if GDC-0084 isable to exert biological effects in tumor tissue. The outcome measurefor this objective was based on the maximum standard uptake value(SUV_(max)) of up to five lesions. The tumor regions of interest (ROIs)were identified for each patient on pretreatment PET imaging andcorresponded to a subset of the target lesions used for analysis of thepatient's pretreatment tumor assessment scans. Determination of PETresponse was done according to the modified European Organization forResearch on the Treatment of Cancer (EORTC) definitions (Young H, BaumR, Cremerius H, et al., “Measurement of clinical and subclinical tumourresponse using [18F]-fluorodeoxy glucose and positron emissiontomograph: review and 1999 EORTC recommendations”, European Organizationfor Research and Treatment of Cancer (EORTC) PET Study Group. Eur JCancer 1999; 35:1773-82). Specifically, the SUV_(max) of each ROI on theon-treatment scans was compared with its SUV_(max) on the correspondingpretreatment scan and the percent change was determined. In the event ofmore than one ROI, the overall percent change in SUV_(max) was thearithmetic mean of the percent changes in SUV_(max) for each of the ROIs(mSUV_(max)). PET response is defined as follows. Progressive disease(PET-PD): percent increase of >20% in mSUV_(max) or the development of anew lesion with an SUV_(max) above background and not explained byanother cause (e.g., infection). Stable disease (PET-SD): percentincrease of ≤20% in mSUV_(max) or a percent decrease of ≤20% inmSUV_(max). Partial response (PET-PR): percent decrease of >20% inmSUV_(max). Complete response (PET-CR): SUV_(max) indistinguishable frombackground in all ROIs (i.e., complete disappearance of all PETlesions).

Tumor response under the RANO guidelines was done generally inaccordance with the Wen method (Wen P Y, Macdonald D R, Reardon D A, etal. “Updated response assessment criteria for high-grade gliomas:Response Assessment in Neuro-Oncology Working Group”, J Clin Oncol 2010;28:1963-72) where the disease is categorized as “complete response”,“partial response”, “stable disease” and “progression”. Among otherfactors, a complete response required all of the following: completedisappearance of all enhancing measurable and non-measurable diseasesustained for at least 4 weeks; no new lesions; and stable or improvednon-enhancing (T2/FLAIR) lesions. A partial response required, amongother factors, ≥50% decrease, compared with baseline, in the sum of theproducts of the perpendicular diameters of all measurable enhancinglesions (such as measured by MRI) sustained for at least 4 weeks; noprogression of non-measurable disease; and no new lesions. Stabledisease occurred if the patient did not qualify for complete response,partial response, or progression. Progression was defined by any of thefollowing: ≥25% increase in the sum of the products of the perpendiculardiameters of all enhancing lesions (compared with the smallest tumormeasurement either at baseline [pretreatment] or after initiation oftherapy [i.e., compared with baseline if no decrease]) on stable orincreasing doses of corticosteroids; a significant increase in T2/FLAIRnonenhancing lesions on stable or increasing doses of corticosteroidscompared with baseline scan or best response after initiation oftherapy, not due to co-morbid events; the appearance of any new lesions;clear progression of non-measurable lesions; or definite clinicaldeterioration not attributable to other causes apart from the tumor, orto a decrease in corticosteroid dose.

Some patients underwent, FDG-PET and additional exploratory MRIassessments to investigate potential pharmacodynamic effects ofGDC-0084. Reduction in ¹⁸F-FDG uptake measured by PET is indicative ofreduced glucose metabolism activity, a likely PD response of PI3Kpathway inhibition. A total of 27 patients underwent FDG-PET imaging atbaseline, cycle 2 day 1 and, for those enrolled on the 45 mg and 65 mgdose levels, also at cycle 1 day 8. On the basis of FDG-PET, five of the27 patients (18.5%) had metabolic partial response according topre-defined criteria. At GDC-0084 doses of at least 45 mg per day, atrend towards decreased median survival in normal brain tissue wasobserved suggesting central nervous system penetration of GDC-0084.GDC-0084 was detected at similar levels in brain tumor and brain tissue,with a brain tissue/tumor to plasma ratio of greater than 1 and greaterthan 0.5 for total drug and free drug, respectively. Of the evaluablepatients, 26 patients (55.3%) had a best overall response of progressivedisease, and 19 patients (40.4%) had stable disease. FDG-PET andconcentration data from brain tumor tissue suggest that GDC-0084 crossesthe blood-brain barrier, with a uniform distribution throughout thebrain.

Thirty-four patients were evaluated by exploratory magnetic resonanceimaging (“MRI”). Dynamic contrast enhanced (DCE) MRI data showed that infour patients with highest drug exposure (AUC_(0-24hr)>8 uMhr) adecrease in tumor Ktrans, a measure of tumor permeability, reflectingtumor angiogenesis, was observed. The Ktrans changes were within thelikely noise range of the measurement (based on variability inlow-exposure cohorts). Overall, none of the MRI derived metrics (Ktrans,Cerebral blood volume, apparent diffusion coefficient) showed anysignificant trend with drug plasma exposure.

Example 26: Transport Assays in Cell Monolayers

Madin-Darby canine kidney (MDCK) cells expressing human P-gp, human BCRPor mouse Bcrp1 and LLC-PK1 cells transfected with mouse P-gp (mdr1a)were used to determine whether GDC-0084 was a substrate of thesetransporters. MDR1-MDCKI cells were licensed from the NCI (NationalCancer Institute, Bethesda, Md.) and Bcrp1-MDCKII, BCRP-MDCKII andMdr1a-LLC-PK1 cells were obtained from the Netherlands Cancer Institute(Amsterdam. The Netherlands). For transport studies, cells were seededon 24-well Millicell plates (Millipore, Billerca, Mass.) 4 days prior touse (polyethylene terephtalate membrane, 1 μm pore size) at a seedingdensity of 2.5×10⁵ cells/mL (except for MDR1-MDCKI, 1.3×10⁵ cells/mL).GDC-0084 was tested at 5 μM in the apical to basolateral (A-B) andbasolateral to apical (B-A) directions. The compound was dissolved intransport buffer consisting of Hank's balanced salt solution (HBSS) with10 mM HEPES (Invitrogen Corporation, Grand Island, N.Y.). Lucifer Yellow(Sigma-Aldrich, St. Louis, Mo.) was used as the paracellular andmonolayer integrity marker. GDC-0084 concentrations in the donor andreceiving compartments were determined by LC-MS/MS analysis. Theapparent permeability (P_(app)), in the apical to A-B and B-Adirections, was calculated after a 2-hour incubation as:P _(app)=(dQ/dt)·(1/AC0)Where: dQ/dt=rate of compound appearance in the receiver compartment;A=Surface area of the insert; C0=Initial substrate concentration at T0.The efflux ratio (ER) was calculated (P_(app, B-A)/P_(app, A-B)).

The results are presented in Table 15 below.

TABLE 15 Apparent Permeability (P_(app)) of GDC-0084 in Transfected CellPapp (10⁻⁶ cm/s) Cell Line A to B B to A P_(app) Ratio MDR1-MDCKI 13.5 ±0.9  11.5 ± 1.6  0.85 ± 0.1  Bcrp1-MDCKII 17.6 ± 2.1  18.6 ± 1.1  1.06 ±0.1  BCRP-MDCKII 23.2 ± 5.4  16.0 ± 1.1  0.71 ± 0.1  Mdr1a-LLC-PK 13.1 ±1.3  19.4 ± 1.3  1.48 ± 0.1 

The apparent permeability (P_(app)) was high and comparable to that ofmetoprolol, the high P_(app) marker used in the same experiments (datanot shown). The efflux ratios (P_(app, B-A)/P_(app, A-B)) did notmarkedly differ from 1 in the MDCK or LLC-PK1 transfected cells,indicating that GDC-0084 was a poor substrate of the efflux transportersP-gp and BCRP.

Example 27: Determination of Plasma Protein and Brain Binding

GDC-0084 protein binding was determined in vitro, in mouse plasma(Bioreclamation, Inc., Hicksville, N.Y.) by equilibrium dialysis using aRED device (Thermo Scientific, Rockford, Ill.), with 300 μL of plasmaand 500 μL of phosphate-buffered saline in the two chambers of thedevice GDC-0084 was added to pooled plasma (n≥3) at a totalconcentration of 5 μM. Plasma samples were equilibrated withphosphate-buffered saline (pH 7.4) at 37° C. in 90% humidity and 5% CO₂for 4 hours. Following dialysis, concentration of GDC-0084 in plasma andbuffer was measured by liquid chromatography-tandem mass spectrometry(LC-MS/MS). The percent GDC-0084 unbound in plasma was determined bydividing the concentration measured in the post-dialysis buffer by thatmeasured in the post-dialysis plasma and multiplying by 100. Incubationswere performed in triplicate. Parameters are presented as mean±standarddeviation.

The free fraction of GDC-0084 in mouse brain was determined as describedby Kalvass et al. (Kalvass J C, Maurer T S, Pollack G M, “Use of plasmaand brain unbound fractions to assess the extent of brain distributionof 34 drugs: comparison of unbound concentration ratios to in vivop-glycoprotein efflux ratios”, Drug Metab. Dispos. 2007; 35(4):660-666).Briefly, brain tissue was homogenized in 3 volumes of phosphate-bufferedsaline and GDC-0084 was added at a final concentration of 5 μM Aliquotsof 300 μl were dialyzed in a RED device (Thermo Scientific, Rockford,Ill.) against a volume of 500 μl buffer for 4 h at 37° C. in anincubator at 90% humidity and 5% CO₂. Following dialysis, tissues andbuffer samples were analyzed as described for the plasma protein bindingstudies.

The results show that GDC-0084 binding to plasma proteins was low, witha free fraction (%) of 29.5±2.7 (n=3) in mouse plasma, when tested at 5μM. Binding to brain tissues was higher, with a free fraction of 6.7%(±1; n=3).

Example 28: Modulation of pAkt and pS6 in the Brain

Inhibition of the PI3K pathway was assessed in the brain of healthy micethrough measurement of two markers, pAkt and pS6. Female CD-1 mice weredosed PO with GDC-0084 at 25 mg/kg. Brains and plasma were collected at1 and 6 hours post-dose, from 3 animals at each time point. Individualbrains were split in half for PD analysis and GDC-0084 concentrationmeasurement. The samples were stored at −80° C. and analyzed forGDC-0084 total concentration. For PD analysis, cell extraction buffer(Invitrogen, Camarillo, Calif.) containing 10 mM Tris pH 7.4, 100 mMNaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1%Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate wassupplemented with phosphatase, protease inhibitors (Sigma, St. Louis,Mo.) and 1 mM PMSF and added to frozen brain biopsies. Brains werehomogenized with a small pestle (Konte Glass Company, Vineland, N.J.),sonicated briefly on ice, and centrifuged at 20,000 g for 20 minutes at4° C. Protein concentration was determined using BCA protein assay(Pierce, Rockford, Ill.). Proteins were separated by electrophoresis andtransferred to NuPage nitrocellulose membranes (Invitrogen, Camarillo,Calif.). Licor Odyssey Infrared detection system (Licor, Lincoln, Nebr.)was used to assess and quantify protein expression. PI3K pathway markerswere evaluated by immunoblotting using antibodies against pAkt^(Ser473),total Akt, pS6^(Ser235/236) and total S6 (Cell Signaling, Danvers,Mass.). The differences in marker levels between the treated and controlmice were evaluated using the Student's t-test (Prism 5, GraphPad).

Following a single oral dose of GDC-0084 (25 mg/kg), pAkt and pS6 levelswere significantly lower than those detected in the control animals(FIG. 23 ). Suppression of pAkt and pS6 reached 90% 1 hour post dose andstayed greater than 70% 6 hours after dosing (FIG. 24 ).

Example 29: U87 and GS2 First Method for Measuring Efficacy in BrainTumor Model

Six female nude mice (Charles River Laboratories) were implanted witheither U87 MGM human glioblastoma cancer cells (described elsewhereherein) or GS2 tumor cells (Gunther H S, Schmidt N O, Phillips H S, etal., “Glioblastoma-derived stem cell-enriched cultures form distinctsubgroups according to molecular and phenotypic criteria”, Oncogene2008; 27(20):2897-2909)), injected via stereotactic surgery into theright striatum (subcortical part of the forebrain) in a volume of 3 to 5μL (250K U87 cells and 100K GS2 cells). A single oral dose of 15 mg/kgGDC-0084 (further comprising 0.5% methylcellulose/0.2% Tween 80 (MCT))was administered 19 to 21 days post-implantation. Mice were euthanizedat 1 and 6 hours post-dose via exsanguination by perfusion underanesthesia. Brains were excised, flash frozen in liquid N₂ and stored ina −80° C. freezer until analyzed. Fresh frozen tissue sections wereobtained on a cryomicrotome (Leica CM3050S, Buffalo Grove, Ill.) at 12μm thickness and thaw-mounted onto indium tin oxide coated glass slides(Bruker Daltonics, Billerica, Mass.). Tissue sections were an analyzedby imaging MALDI MS, providing signal intensities (and not absolutequantitation), followed by cresyl violet staining for histologicalinterrogation.

For MALDI MAS analysis, a 40 mg/mL solution of 2,5-dihydroxybenzoic acid(Sigma-Aldrich, St. Louis, Mo.) was prepared in methanol:water (70:30v/v). A stable-labeled internal standard, [D6]GDC-0084, was spiked intothe MALDI matrix solution at 2 μM prior to deposition onto the tissuesections. Matrix solution was homogenously spray-coated onto the tissueusing a HTX TM-Sprayer (HTX technologies. Chapel Hill, N.C.).Matrix-coated tissue sections were transferred to the MALDI massspectrometer (SolariX 7T FT-ICR, Bruker Daltonics, Bremen, Germany) forimaging analysis. Imaging data were collected at 100 μm pixel resolutionin positive ionization mode, under continuous accumulation of selectedions (CASI) windows optimized for a 50 Da window centered on m z 383 (mz 358-408). Laser intensity and number of shots were optimized forsensitivity of the parent drug (1200 shots) with ion detection collectedover the mass range of m/z 150-3000. Drug images were generated based onaccurate mass the parent drug (GDC-0084 m/z 383.1938) using FlexImagingv4.0 64-bit (Bruker Daltonics, Billerica, Mass.) with a mass toleranceof ±2 mDa and normalized to internal standard response.

Following completion of the imaging experiments, matrix coaling wasremoved by rinsing the glass slide in 100% methanol for 30 seconds oruntil the entire matrix was visibly removed. Tissue sections werestained utilizing a freshly prepared 0.5% cresyl violet stainingsolution (Chaurand P, Schwartz S A, Billheimer D, Xu B J, Crecelius A,Caprioli R M, “Integrating histology and imaging mass spectrometry”,Anal Chem. 2004; 76(4): 1145-1155) by submerging the glass slide for 30seconds, then rinsed for an additional 30 seconds in two cycles of 100%ethanol. Microscope images were obtained on an Olympus BX51 (Tokyo,Japan) at 10× magnification and stitched using MicroSuite Analyticalv3.0 software (Olympus, Tokyo, Japan). Subsequently, stained images wereco-registered to the optical images in FlexImaging for visualization andannotation of tumor and non-tumor regions for the drug images.

To assess drug distribution, imaging MALDI MS data from U87 and GS2tumor models were co-registered to the cresyl violet stained microscopeimages in FlexImaging Regions of interest (ROIs) that were selectedbased on the anatomical features defined in the histological imageincluding tumor and non-tumor regions. Drug intensity for each pixelwithin the defined ROI was extracted and exported. Drug intensities werebinned in 0.1 increments over a range of 0.0 to 2.0. Histogram plotswere created in GraphPad Prism 5 to visualize the distribution of pixelintensity frequencies.

Example 30: U87 and GS2 Second Method for Measuring Efficacy in BrainTumor Model

U87 glioblastoma cancer cells (described elsewhere herein) and GS2glioblastoma cells were selected to test the efficacy of GDC-0084 in amouse brain model. These U87 and GS2 models are PTEN-deficient, with theGS2 cell line presenting a copy number loss at the PTEN locus (Gunther HS, et al.) with no detectable PTEN protein by western blot (Carlson B L,Pokomy J L, Schroeder M A, Sarkaria J N., “Establishment, maintenanceand in vitro and in vivo applications of primary human glioblastomamultiforme (GBM) xenograft models for translational biology studies anddrug discovery”, Curr Protoc Pharmacol. 2011; Chapter 14: Unit 14 16).The identity of the two cell lines was confirmed by STR profiling (DNADiagnostics Center) using cells within 5 passages of those utilized forin vivo studies. The U87 (250K) and GS2 (100K) tumor cells were injectedvia stereotaxic surgery into the right striatum (subcortical part of theforebrain) in a volume of 3-5 μl. For each experiment, mice wererandomized into groups of 10 to obtain comparable mean tumor volumesbetween treatment and control groups for each model. Treatments wereadministered GDC-0084 (15 mg/kg), or vehicle (MCT) PO daily for 2 or 4weeks, respectively, starting 7 days (U87) or 14 days (GS2) post tumorcell inoculation. Mouse body weights were recorded twice per week duringthe study and animals were euthanized if body weight loss was greaterthan 20% from their initial body weight. Tumor volumes were monitored byex vivo micro micro-computed tomography (micro-CT) imaging and T2 MRIfor the GBM models U87 and GS2, respectively. The differences betweentreatment groups were evaluated using Student's t test in Prism (Prism5, GraphPad). MRI was performed on a Varian 9.4T MRI system with a 30 mmquadrature volume coil. During the imaging, animals were kept underanesthesia with 2% isoflurane in air. Body temperature was continuouslymonitored using a rectal probe and was maintained at 37° C. by aheated-air flow system regulated by in-house LabVIEW controllersoftware. A T2-weighted fast spin echo, multi-slice (FSEMS) sequence wasused to detect lesions by MRI. 12-20 axial 0.5-0.8 mm-thick slices wereacquired with a 20×20 mm field of view (FOV), and 128×128 matrix,zero-filled to 256×256 mages. TR=3500-4000 ms, TE=9-10 ms, ETL=8,k-zero=4, NEX=8. Tumor volumes were calculated from the T2-weightedFSEMS images using an intensity threshold based region growing tool inMRVision software. Brain sample preparation, micro-CT scanning, andimage analysis for ex-vivo micro-CT imaging were performed as describedpreviously (de Crespigny A, Bou-Reslan H, Nishimura M C, Phillips H,Carano R A, D'Arceuil H E, “3D micro-CT imaging of the postmortembrain”, J. Neurosci. Methods. 2008; 171 (2): 207-213).

In the studies conducted with the GS2 tumor-bearing mice, plasma andbrains were also collected at the end of treatment to measure GDC-0084and assess PI3K pathway modulation in the tumor. Each brain wasdissected to separate the tumor from the healthy tissues. Plasma andnormal brains were processed and analyzed by LC-MS/MS. The GS2 tumorsisolated from the brains were processed and the PI3K pathway markerspAkt, pS6 and p4EBP1 were measured as described previously.

Example 31: MALDI Imaging Results

Distribution of GDC-0084 in the brain and intracranial U87 and GS2tumors following administration of a single PO dose (15 mg/kg) wasinvestigated by MALDI imaging. Brains were collected 1 hour post doseand images presented in FIGS. 25A and 25B show that GDC-0084 distributedreadily and quite evenly throughout the brain, including in the GS2(FIG. 25A) and U87 (FIG. 25B) tumors. In addition, the homogeneity andpattern of distributions of GDC-0084 in the tumors and non-tumoredregions of the brains were further analyzed. The frequency of signalintensities (frequency of pixel intensities) appeared to follow a normaldistribution in healthy brain, superimposed (mean pixel intensity 0.54)to that observed in U87 tumors (FIG. 26A); mean pixel intensity 0.54). AGaussian distribution of signals was also observed in GS2 tumors (FIG.26B), with however a slightly lower mean in pixel intensity (0.34 vs.0.55), suggesting an overall lower GDC-0084 concentration in GS2 tumorsthan in normal brain. Comparisons of the GDC-0084 signal homogeneity innon-tumored brain regions between the U87 and GS2 tumor-bearing miceshowed identical distribution (FIG. 26C), confirming the reproducibleand consistent brain penetration properties of GDC-0084. Similar resultswere obtained in brains collected at 6 hours post dose. Furthermore, tocontrast the distribution of GDC-0084 to that of a non-brain penetrantcompound, MALDI images previously obtained with pictilisib in the U87tumor model (Salphati L, Shahidi-Latham S, Quiason C, et al.,“Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib(GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastomamodels-assessment by matrix-assisted laser desorption ionizationimaging”, Drug Metab Dispos. 2014; 42(7):1110-1116) were re-analyzedusing the approach utilized here. In comparative analysis, reanalysis ofdata previously obtained with the non-brain penetrant compound pictilisb(Salphati, et al.) showed heterogeneous (non Gaussian) intra-tumordistribution of pixel intensities (FIG. 26D). While signal intensitiesin the U87 tumor for GDC-0084 could be fit to a Gaussian curve, signalsfrom pictilisib were concentrated in the low intensity bins, with adistribution that appeared more heterogeneous (FIG. 26D). As compared topictilisib, GDC-0084 provided for improved homogeneous andundifferentiated compound distribution throughout healthy brain tissueand tumor tissue. Based on the brain tumor model results, it is believedthat GDC-0084 provides for improved treatment, not only the core of thetumor, but also invasive glioma cells protected by an intact BBB orblood-tumor barrier.

Example 32: Brain Tumor Model Results

The efficacy of GDC-0084 was tested in the U87 and GS2 intracranialmodels. GDC-0084 was administered PO at 15 mg/kg daily for 2 and 4 weeksto U87 and GS2 tumor-bearing mice, respectively. The effect of thetreatment on the U87 and GS2 tumor volumes was assessed at the end ofthe dosing period. Images of U87 tumor obtained by micro-CT arepresented in FIG. 27A. The U87 tumor volumes were reduced byapproximately 70%, when compared to the vehicle control, (FIG. 27B)following treatment with GDC-0084. Similarly, the GS2 tumors measured byMRI (FIG. 27C) in the treated mice were significantly (p<0.01) smaller(≈40%) than those in the control group (FIG. 27D). Plasma and healthybrain concentrations of GDC-0084 were measured at the end of the studyin the GS2 tumor-bearing mice and are presented along withbrain-to-plasma ratios in Table 16 below. Brain concentrations in thenormal part of the brain and brain-to-plasma ratios were comparable tothose obtained previously (Table 9). Modulation of the PI3K pathway inthe GS2 tumors was assessed by western blot at the end of the dosingperiod, 2 and 8 hours after the final administration of GDC-0084 (FIG.28A). Levels of pAkt were significantly reduced at 2 and 8 hours, by 90and 70%, respectively. Suppression of pS6 and p4EBP1 was less pronouncedat 2 hours, reaching 35 and 43%, respectively. These two markers wereback to baseline levels 8 hours post-dose (FIG. 28B)

TABLE 16 Plasma Concentrations, Brain Concentrations and Brain-to-PlasmaRatio Measured 2 and 8 hours Following PO Administration of GDC-0084 (15mg/kg) to GS2 Tumor-Bearing Mice (non-tumored half of the brain) Timepost- Brain-to- dose (h) Brain (μM) Plasma (μM) Plasma Ratio 2 5.51 ±1.58 3.64 ± 2.05 1.67 ± 0.51 8 2.48 ± 1.25 2.01 ± 1.19 1.29 ± 0.16

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

EMBODIMENTS

-   A. A process for preparing a compound of Formula III from a compound    of Formula II in a reaction mixture according to the following    reaction scheme:

-   -   the process comprising:        -   (i) forming a reaction mixture comprising the compound            Formula II, organoboron-R⁴, the solvent system comprising at            least 5 v/v % water, the base and the catalyst;        -   (ii) reacting the reaction mixture at a temperature of less            than 100° C. to form a reaction product mixture comprising            compound Formula III; and        -   (iii) isolating the compound Formula III, a stereoisomer,            geometric isomer, tautomer, or a pharmaceutically acceptable            salt thereof, from the reaction product mixture,    -   wherein        -   the catalyst comprises palladium and the reaction mixture            comprises less than 0.05 equivalents of catalyst per            equivalent of compound Formula II;        -   X¹ is S, O, N, NR⁶, CR¹, C(R¹)₂, or —C(R¹)₂O—;        -   X² is C, CR² or N;        -   X³ is C, CR³ or N;        -   X⁴ is halogen;        -   A is a 5, 6, or 7-membered carbocyclyl or heterocyclyl ring            fused to X² and X³, optionally substituted with one or more            R⁵, R¹⁰ or R¹⁵ groups;        -   R⁶ is H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,            —(C₁-C₁₂ alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂            alkylene)(—C₂-C₂₀ heterocyclyl), —(C₁-C₁₂            alkylene)-C(═O)—(C₂-C₂₀ heterocyclyl), (C₃-C₁₂            alkylene)-(C₆-C₂₀ aryl), and —(C₁-C₁₂ alkylene)-(C₁-C₂₀            heteroaryl), where alkyl, alkenyl, alkynyl, alkylene,            carbocyclyl, heterocyclyl, aryl, and heteroaryl are            optionally substituted with one or more groups independently            selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH,            —CH₂CH₂OH, —(CH₃)₂OH, —CH₂OCH₃, —CN, —CO₂H, —COCH₃,            —COC(CH₃)₃, —CO₂CH₃, —CONH₂, —CONHCH₃, —CON(CH₃)₂,            —C(CH₃)₂CONH₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCOCH₃,            —NHS(O)₂CH₃, —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O,            —OH, —OCH₃, —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl,            cyclobutyl, oxetanyl, morpholino, and            1,1-dioxo-thiopyran-4-yl;        -   R¹, R², and R³ are independently selected from H, F, Cl, Br,            I, —CH₃, —CH₂CH₃, —C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH,            —CH₂OCH₃, —CN, —CF₃, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃,            —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂, —CONH₂, —NO₂, —NH₂,            —NHCH₃, —N(CH₃)₂, —NHCOCH₃, —NHS(O)₂CH₃,            —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,            —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl,            oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl;        -   R⁴ is selected from C₆-C₂₀ aryl, C₂-C₂₀ heterocyclyl and            C₁-C₂₀ heteroaryl, each of which are optionally substituted            with one or more groups independently selected from F, Cl,            Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃,            —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂, CONH(CH₃),            —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃,            —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃,            —NHS(O)₂CH₃, —N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl,            benzyloxy, morpholinyl, morpholinomethyl, and            4-methylpiperazin-1-yl;        -   Each R⁵, R¹⁰ and R¹⁵ is independently selected from C₁-C₁₂            alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, —(C₄-C₁₂            alkylene)-(C₃-C₁₂ carbocyclyl), —(C₁-C₁₂ alkylene)-(C₂-C₂₀            heterocyclyl), —(C₄-C₁₂ alkylene)-C(O)—(C₂-C₂₀            heterocyclyl), —(C₄-C₁₂ alkylene)-(C₆-C₂₀ aryl), and            —(C₁-C₁₂ alkylene)-(C₁-C₂₀ heteroaryl); or two geminal R⁵,            R¹⁰ and/or R¹⁵ groups form a 3, 4, 5, or 6-membered            carbocyclyl or heterocyclyl ring, where alkyl, alkenyl,            alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl, and            heteroaryl are optionally substituted with one or more            groups independently selected from F, Cl, Br, I, —CH₃,            —CH₂CH₃, —C(CH₃)₃, —CH₂OH, —CH₂CH₂OH, —C(CH₃)₂OH, —CH₂OCH₃,            —CN, —CH₂F, —CHF₂, —CF₃, —CO₂H, —COCH₃, —COC(CH₃)₃, —CO₂CH₃,            —CONH₂, —CONHCH₃, —CON(CH₃)₂, —C(CH₃)₂CONH₂, —NO₂, —NH₂,            —NHCH₃, —N(CH₃)₂, —NH—COCH₃, —NHS(O)₂CH₃,            —N(CH₃)C(CH₃)₂CONH₂, —N(CH₃)CH₂CH₂S(O)₂CH₃, ═O, —OH, —OCH₃,            —S(O)₂N(CH₃)₂, —SCH₃, —S(O)₂CH₃, cyclopropyl, cyclobutyl,            oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl; and        -   mor is selected from:

-   -   -   wherein mor is optionally substituted with one or more R⁷            groups independently selected from F, Cl, Br, I, —CH₃,            —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂,            —CN, —CF₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH, —CH₂C(CH₃)₂OH,            —CH(CH₃)OH, —CH(CH₂CH₃)OH, —CH₂CH(OH)CH_(3J)—C(CH₃)₂OH,            —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂, —CH(CH₂CH₃)F,            —C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃,            —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃,            —NHCH(CH₃)₂, —NHCH₂CH₂OH, —NHCH₂CH₂OCH₃, —NHCOCH₃,            —NHCOCH₂CH₃, —NHCOCH₂OH, —NHS(O)₂CH₃, —N(CH₃)S(O)₂CH₃, ═O,            —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃,            —NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃,            —S(O)₂NH₂, —S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃.

-   A1. The process of embodiment A wherein the solvent system further    comprises at least one polar aprotic solvent selected from    N-methylpyrrolidone, methyl isobutyl ketone, methyl ethyl ketone,    tetrahydrofuran, dichloromethane, ethyl acetate, acetone,    N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

-   A2. The process of embodiment A1 wherein the ratio of water to the    at least one polar aprotic solvent is from about 1:10 v/v to about    5:1 v/v, from about 1:1 v/v to about 1:10 v/v, or from about 1:3 v/v    to about 1:7 v/v.

-   A3. The process of embodiment A1 or A2 wherein the solvent system    comprises water and tetrahydrofuran.

-   A4. The process of any one of embodiments A1 to A3 wherein the    solvent system consists essentially of water and the at least one    polar aprotic solvent.

-   A5. The process of any one of embodiments A to A4 wherein the    organoboron-R⁴ is 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2    yl)-R⁴.

-   A6. The process of any one of embodiments A to A5 wherein the base    is selected from K₃PO₄, Cs₂CO₃, and KOH.

-   A7. The process of any one of embodiments A to A6 wherein the base    is K₃PO₄.

-   A8. The process of any one of embodiments A to A7 wherein the    equivalent ratio of base to compound Formula II is at least 1:1,    from about 1:1 to about 3:1, or about 2:1.

-   A9. The process of any one of embodiments A to A8 wherein the    catalyst comprising palladium is selected from    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    (“PdXphos”); 1,1′-bis(diphenylphosphino)ferrocene]    dichloropalladium(II) complex with dichloromethane (“PdCl₂ dppf    CH₂Cl₂”);    Bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II)    (“Pd(amphos)Cl₂”);    dichlorobis(di-tert-butylphenylphosphine)palladium(II) (“Pd 122”);    PdCl₂(PPh₃)₂; Pd(r-Bu)₃; Pd(PPh₃)₄; Pd(Oac)/PPh₃; Cl₂Pd[(Pet₃)]₂;    Pd(DIPHOS)₂; Cl₂Pd(Bipy); [PdCl(Ph₂PCH₂PPh₂)]₂; Cl₂Pd[P(o-tol)₃]₂;    Pd₂(dba)₃/P(o-tol)₃; Pd₂(dba)/P(furyl)₃; Cl₂Pd[P(furyl)₃]₂;    Cl₂Pd(PmePh₂)₂; Cl₂Pd[P(4-F-Ph)₃]₂; Cl₂Pd[P(C₆F₆)₃]₂;    Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂; Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂; palladium    acetate, microencapsulated in a polyuria matrix, comprising 0.4    mmol/g Pd; palladium acetate and triphenylphosphine,    microencapsulated in a polyuria matrix, comprising 0.4 mmol/g Pd and    0.3 mmol/g phosphorous; and palladium acetate and BINAP,    microencapsulated in a polyuria matrix, comprising 0.4 mmol/g Pd.

-   A10. The process of embodiment A9 wherein the catalyst comprising    palladium is selected from    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    and 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)    complex with dichloromethane, or is    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II).

-   A11. The process of any one of embodiments A to A10 wherein the    equivalent ratio of the catalyst comprising palladium to compound    Formula II is between about 0.003:1 and 0.05:1, from about 0.003:1    to about 0.03:1 or from about 0.004:1 to about 0.02:1.

-   A12. The process of any one of embodiments A to A11 wherein the    catalyst is    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    and the equivalent ratio of the catalyst comprising palladium to    compound Formula II is from about 0.004:1 to about 0.015:1, from    about 0.004:1 to about 0.01:1, from about 0.004:1 to about 0.007:1,    or about 0.005:1.

-   A13. The process of any one of embodiments A to A11 wherein the    catalyst is    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    or 1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II)    complex with dichloromethane and the equivalent ratio of the    catalyst comprising palladium to compound Formula II is from about    0.005:1 to about 0.04:1, from about 0.005:1 to about 0.03:1, from    about 0.01:1 to about 0.03:1, or about 0.02:1.

-   A14. The process of any one of embodiments A to A13 wherein the    reaction temperature is between about 40° C. and 100° C., from about    40° C. to about 90° C., from about 40° C. to about 80° C., from    about 50° C. to about 80° C. or from about 55° C. to about 75° C.

-   A15. The process of any one of embodiments A to A14 further    comprising adding a polar protic solvent to the reaction product    mixture to form an admixture comprising greater than 25 v/v % water    and separating compound Formula III from the reaction product    mixture by solid liquid separation.

-   A16. The process of embodiment A15 wherein the polar protic solvent    is selected from water, methanol, ethanol, isopropanol, n-propanol,    and acetic acid.

-   A17. The process of embodiment A16 wherein the polar protic solvent    is water.

-   A18. The process of embodiment A17 wherein the volume ratio of the    solvent system to water added to the reaction product mixture is    from about 1:5 v/v to about 5:1 v/v, from about 1:3 v/v to about 3:1    v/v, from about 1:2 v/v to about 2:1 v/v, from about 1:1.5 v/v to    about 1.5:1 v/v, or about 1:1 v/v.

-   A19. The process of embodiment A17 or embodiment A18 further    comprising adding compound Formula III seed crystals to admixture of    the reaction product mixture and water.

-   A20. The process of any one of embodiments A to A19 further    comprising a purification step comprising:    -   (i) admixing compound Formula III with a metal scavenger in a        solvent system comprising at least one polar protic solvent;    -   (ii) heating the admixture to dissolve compound Formula III;    -   (iii) filtering the heated admixture;    -   (iv) reducing the temperature of the filtrate and admixing        compound Formula III seed crystals with the cooled filtrate;    -   (v) reducing the temperature of the admixture of filtrate and        seed crystals to induce crystallization of purified compound        Formula III; and    -   (vi) collecting purified compound Formula III crystals.

-   A21. The process of embodiment A20 wherein:    -   (i) the solvent system comprises water and acetic acid or        consists essentially of water and acetic acid wherein the volume        ratio of acetic acid to water is from about 1:1 to about 10:1,        from about 1:1 to about 5:1 or from about 1:1 to about 3:1, or        about 3:1;    -   (ii) the metal scavenger is silica-thiol; and    -   (iii) the dissolution temperature is from about 80° C. to about        100° C., the seed crystals are combined with the filtrate at a        temperature of from about 70° C. to about 80° C., and the        crystallization temperature is from about 0° C. to about 10° C.

-   A22. The process of any one of embodiments A to A21 wherein the    yield of compound Formula III is at least 75%, at least 80% at least    85% or at least 90%.

-   A23. The process of any one of embodiments A to A22 wherein the    purity of compound Formula III is at least 97%, at least 97.5%, or    at least 98% (area % as determined by HPLC).

-   A24. The process of any one of embodiments A to A23 wherein X¹ is N,    NR⁶, CR¹, C(R¹)₂ or C(R¹)₂O and X³ is C or CR³.

-   A25. The process of embodiment A24 wherein X¹ and X² are N, and X³    is C.

-   A26. The process of any one of embodiments A to A25 wherein A is an    optionally substituted 6-membered heterocycle comprising at least    one heteroatom selected from N and O.

-   A27. The process of embodiment A26 wherein X² is N and A is    optionally substituted morpholine.

-   A28. The process of any one of embodiments A to A27 wherein mor is    optionally substituted morpholine

-   A29. The process of any one of embodiments A to A28 wherein R⁴ is    selected from optionally substituted C₆ aryl, optionally substituted    C₆ heterocycle and optionally substituted C⁶ heteroaryl.

-   A30. The process of embodiment A29 wherein R⁴ is optionally    substituted 0, heteroaryl comprising one or two N heteroatoms.

-   A31. The process of embodiment A30 wherein R⁴ is optionally    substituted pyrimidine.

-   A32. The process of any one of embodiments A to A31 wherein compound    Formula III is

-   B. A process for preparing a compound of Formula IIa from a compound    of Formula I in a reaction mixture according to the following    reaction scheme:

-   -   the process comprising:    -   (i) forming a reaction mixture comprising compound Formula I,        organic halide, a solvent system, a phase transfer catalyst, and        a base, (ii) reacting the reaction mixture to form a reaction        product mixture comprising compound Formula IIa, a stereoisomer,        geometric isomer, tautomer, or a pharmaceutically acceptable        salt thereof, and (iii) isolating compound Formula IIa from the        reaction product mixture,    -   wherein        -   the solvent system comprises at least 5 v/v % water;        -   X is a halide;        -   Each R⁵, R¹⁰ and R¹⁵ are independently selected from H,            C₁-C₁₀ hydrocarbyl or from C₁-C₅ hydrocarbyl, wherein each            hydrocarbyl is optionally substituted, two geminal R⁵, R¹⁰            and/or R¹⁵ groups are oxo, or two geminal R⁵, R¹⁰ and/or R¹⁵            groups form a 3, 4, 5, 6, or 7-membered carbocyclyl or            heterocyclyl, wherein the carbocyclyl or heterocyclyl is            optionally substituted;        -   mor is selected from:

-   -   -   wherein mor is optionally substituted with one or more R⁷            groups independently selected from F, Cl, Br, I, —CH₃,            —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂,            —CN, —CF₃, —CH₂OH, —CH₂OCH₃, —CH₂CH₂OH, —CH₂C(CH₃)₂OH,            —CH(CH₃)OH, —CH(CH₂CH₃)OH, —CH₂CH(OH)CH₃, —C(CH₃)₂OH,            —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂, —CH(CH₂CH₃)F,            —C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃,            —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃,            —NHCH(CH₃)₂, —NHCH₂CH₂OH, —NHCH₂CH₂OCH₃, —NHCOCH₃,            —NHCOCH₂CH₃, —NHCOCH₂OH, —NHS(O)₂CH₃, —N(CH₃)S(O)₂CH₃, ═O,            —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃,            —NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃,            —S(O)₂NH₂, —S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃; and        -   wherein in formula I R²⁰ is —OH or —NHR²¹ wherein R²¹ is as            defined for R⁵, and wherein in formula IIa R²⁰ is —O— or            —NR²¹—.

-   B1. The process of embodiment B wherein the solvent system comprises    at least 50 v/v % water, at least 75 v/v % water, at least 90 v/v %    water, or consists essentially of water.

-   B2. The process of embodiment B or embodiment B1 wherein the base is    selected from K₃PO₄, Cs₂CO₃, K₂CO₃, KOAc, NaOAc, Na₂CO₃ and KOH.

-   B3. The process of embodiment B2 wherein the base is KOH.

-   B4. The process of any one of embodiments B to B3 wherein the phase    transfer catalyst is selected from a quaternary ammonium salt and a    phosphonium salt.

-   B5. The process of embodiment B4 wherein the phase transfer catalyst    is selected from tetra-n-butylammonium bromide,    benzyltrimethylammonium chloride, benzyltriethyl ammonium chloride,    methyltricaprylammonium chloride, methyltributylammonium chloride,    and methyltrioctylammonium chloride.

-   B6. The process of embodiment B5 wherein the phase transfer catalyst    is tetra-n-butylammonium bromide.

-   B7. The process of any one of embodiments B to B6 wherein the molar    ratio of the organic halide dibromoethane to compound Formula I is    from greater than 2:1 to about 4:1, between 2:1 and about 4:1, or    about 3:1.

-   B8. The process of any one of embodiments B to B7 wherein the    organic halide and the base are present in about equimolar amounts.

-   B9. The process of any one of embodiments B to B8 wherein the    reaction temperature is from about 40° C. to about 90° C., from    about 40° C. to about 70° C., from about 40° C. to about 60° C., or    about 50° C.

-   B10. The process of any one of embodiments B to B9 further    comprising admixing a polar protic solvent with the reaction product    mixture followed by reducing the temperature of the admixture to    induce crystallization of compound Formula IIa in the reaction    product mixture, wherein the crystallized compound Formula IIa is    isolated from the reaction product mixture.

-   B11. The process of embodiment B10 wherein the polar protic solvent    is selected from water, methanol, ethanol, isopropanol, n-propanol,    and acetic acid.

-   B12. The process of embodiment B11 wherein the polar protic solvent    is ethanol.

-   B13. The process of embodiment B12 wherein volume ratio of the    solvent system to ethanol is from about 1:5 v/v to about 5:1 v/v,    from about 1:3 v/v to about 3:1 v/v, from about 1:2 v/v to about 2:1    v/v, from about 1:1 v/v to about 1:2 v/v, or about 1:1.3 v/v.

-   B14. The process of embodiment B12 or B13 further comprising adding    compound Formula IIa seed crystals to the admixture of the reaction    product mixture and ethanol.

-   B15. The process of any one of embodiments B to B14 wherein the    yield of compound Formula II is at least 60%, at least 65% at least    70% or at least 75%.

-   B16. The process of any one of embodiments B to B15 wherein the    purity of compound Formula II is at least 97%, at least 97.5%, at    least 98%, at least 98.5% or at least 99% (area % as determined by    HPLC.

-   B17. The process of any one of embodiments B to B16 wherein each R⁵,    R¹⁰ and R¹⁵ is independently selected from H and optionally    substituted C₁₋₅ alkyl, or two geminal R⁵, R¹⁰ and/or R¹⁵ groups    together are oxo or form a 3 to 6-membered cycloalkyl or    heterocycloalkyl having one or two hetero atoms selected from N and    O.

-   B18. The process of embodiment B17 wherein each R⁵, R¹⁰ and R¹⁵ is    independently selected from H, C₁₋₅ alkyl and C₁₋₅ alkyl substituted    with at least one of deuterium, halogen and hydroxyl.

-   B19. The process of any one of embodiments B to B18 wherein in    formula I R²⁰ is —OH, —NH₂ or —NH—C₁₋₅ alkyl.

-   B20. The process of embodiment B19 wherein in formula I R²⁰ is —OH.

-   B21. The process of any one of embodiments B to B20 wherein the    organic halide is 1,2-dibromoethane.

-   B22. The process of any one of embodiments B to B21 wherein compound    Formula IIa is

-   C. The process of any one of embodiments B to B22 further comprising    preparing a compound of Formula IIIa from a compound of Formula IIa    in a reaction mixture according to the following reaction scheme:

-   -   the process comprising:        -   (i) forming a reaction mixture comprising compound Formula            IIa, organoboron-R⁴, the solvent system comprising at least            5 v/v % water, the base and the catalyst;        -   (ii) reacting the reaction mixture to form a reaction            product mixture comprising compound Formula IIIa; and        -   (iii) isolating compound Formula IIIa, a stereoisomer,            geometric isomer, tautomer, or a pharmaceutically acceptable            salt thereof, from the reaction product mixture by solid            liquid separation wherein the yield of compound Formula IIIa            is at least 75%,    -   wherein        -   the catalyst comprises palladium and the reaction mixture            comprises less than 0.05 equivalents of catalyst per            equivalent of compound Formula IIa; and        -   R⁴ is selected from C₆-C₂₀ aryl, C₂-C₂₀ heterocyclyl and            C₁-C₂₀ heteroaryl, each of which are optionally substituted            with one or more groups independently selected from F, Cl,            Br, I, —CH₃, —CH₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃,            —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H, —CONH₂, CONH(CH₃),            —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃,            —OCH₂CH_(3J)—OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃,            —NHS(O)₂CH₃, —N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl,            benzyloxy, morpholinyl, morpholinomethyl, and            4-methylpiperazin-yl.

-   C1. The process of embodiment C wherein the solvent system further    comprises at least one polar aprotic solvent selected from    tetrahydrofuran, dichloromethane, ethyl acetate, acetone,    N,N-dimethylformamide, acetonitrile and dimethyl sulfoxide.

-   C2. The process of embodiment C1 wherein the ratio of water to the    at least one polar aprotic solvent is from about 1:10 v/v to about    5:1 v/v, from about 1:1 v/v to about 1:10 v/v, or from about 1:3 v/v    to about 1:7 v/v.

-   C3. The process of embodiment C1 or C2 wherein the solvent system    comprises water and tetrahydrofuran.

-   C4. The process of any one of embodiments C1 to C3 wherein the    solvent system consists essentially of water and the at least one    polar aprotic solvent.

-   C5. The process of any one of embodiments C to C4 wherein the    organoboron-R⁴ is 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2    yl)-R⁴.

-   C6. The process of any one of embodiments C to C5 wherein the base    is selected from K₃PO₄, Cs₂CO₃, and KOH.

-   C7. The process of any one of embodiments C to C6 wherein the base    is K₃PO₄.

-   C8. The process of any one of embodiments C to C7 wherein the    equivalent ratio of base to compound Formula IIa is at least 1:1,    from about 1:1 to about 3:1, or about 2:1.

-   C9. The process of any one of embodiments C to C8 wherein the    catalyst comprising palladium is selected from    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    (“PdXphos”); 1,1′-bis(diphenylphosphino)ferrocene]    dichloropalladium(II) complex with dichloromethane (“PdCl₂ dppf    CH₂Cl₂”);    Bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II)    (“Pd(amphos)Cl₂”);    dichlorobis(di-tert-butylphenylphosphine)palladium(II) (“Pd 122”);    PdCl₂(PPh₃)₂; Pd(t-Bu)₃; Pd(PPh₃)₄; Pd(Oac)/PPh₃; Cl₂Pd[(Pet₃)]₂;    Pd(DIPHOS)₂; Cl₂Pd(Bipy); [PdCl(Ph₂PCH₂PPh₂)]₂; Cl₂Pd[P(o-tol)₃]₂;    Pd₂(dba)₃/P(o-tol)₃; Pd₂(dba)/P(furyl)₃; Cl₂Pd[P(furyl)₃]₂;    Cl₂Pd(PmePh₂)₂; Cl₂Pd[P(4-F-Ph)₃]₂; Cl₂Pd[P(C₆F₆)₃]₂;    Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂; Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂; palladium    acetate, microencapsulated in a polyuria matrix, comprising 0.4    mmol/g Pd; palladium acetate and triphenylphosphine,    microencapsulated in a polyuria matrix, comprising 0.4 mmol/g Pd and    0.3 mmol/g phosphorous; and palladium acetate and BINAP,    microencapsulated in a polyuria matrix, comprising 0.4 mmol/g Pd.

-   C10. The process of embodiment C9 wherein the catalyst comprising    palladium is selected from    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    and 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)    complex with dichloromethane.

-   C11. The process of any one of embodiments C to C10 wherein the    equivalent ratio of the catalyst comprising palladium to compound    Formula IIa is between about 0.003:1 and 0.05:1, from about 0.003:1    to about 0.03:1 or from about 0.004:1 to about 0.02:1.

-   C12. The process of any one of embodiments C to C11 wherein the    catalyst is    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    and the equivalent ratio of the catalyst comprising palladium to    compound Formula IIa is from about 0.004:1 to about 0.015:1, from    about 0.004:1 to about 0.01:1, from about 0.004:1 to about 0.007:1,    or about 0.005:1.

-   C13. The process of any one of embodiments C to C11 wherein the    catalyst is    chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II)    and 1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)    complex with dichloromethane and the equivalent ratio of the    catalyst comprising palladium to compound Formula IIa is from about    0.005:1 to about 0.04:1, from about 0.005:1 to about 0.03:1, from    about 0.01:1 to about 0.03:1, or about 0.02:1.

-   C14. The process of any one of embodiments C to C13 further    comprising adding a polar protic solvent to the reaction product    mixture to form an admixture comprising at least 25 v/v % water and    separating compound Formula IIIa from the reaction product mixture.

-   C15. The process of embodiment C14 wherein the polar protic solvent    is selected from water, methanol, ethanol, isopropanol, n-propanol,    and acetic acid.

-   C16. The process of embodiment C15 wherein the polar protic solvent    is water.

-   C17. The process of embodiment C16 wherein the volume ratio of the    solvent system to water added to the reaction product mixture is    from about 1:5 v/v to about 5:1 v/v, from about 1:3 v/v to about 3:1    v/v, from about 1:2 v/v to about 2:1 v/v, from about 1:1.5 v/v to    about 1.5:1 v/v, or about 1:1 v/v.

-   C18. The process of embodiment C16 or C17 further comprising adding    compound Formula IIIa seed crystals to admixture of the reaction    product mixture and water.

-   C19. The process of any one of embodiments C to C18 further    comprising a purification step comprising:    -   (i) admixing compound Formula IIIa with a metal scavenger in a        solvent system comprising at least one polar protic solvent;    -   (ii) heating the admixture to dissolve compound Formula IIIa;    -   (iii) filtering the heated admixture;    -   (iv) reducing the temperature of the filtrate and admixing        compound Formula IIIa seed crystals with the cooled filtrate;    -   (v) reducing the temperature of the admixture of filtrate and        seed crystals to induce crystallization of purified compound        Formula IIIa; and    -   (vi) collecting purified compound Formula IIIa crystals.

-   C20. The process of embodiment C19 wherein:    -   (i) the solvent system comprises water and acetic acid or        consists essentially of water and acetic acid wherein the volume        ratio of acetic acid to water is from about 1:1 to about 10:1,        from about 1:1 to about 5:1 or from about 1:1 to about 3:1, or        about 3:1;    -   (ii) the metal scavenger is silica-thiol; and    -   (iii) the dissolution temperature is from about 80° C. to about        100° C., the seed crystals are combined with the filtrate at a        temperature of from about 70° C. to about 80° C., and the        crystallization temperature is from about 0° C. to about 10° C.

-   C21. The process of any one of embodiments C to C20 wherein the    yield of compound Formula IIIa based on compound Formula IIa is at    least 75%, at least 80% at least 85% or at least 90%.

-   C22. The process of any one of embodiments C to C21 wherein the    purity of compound Formula IIIa is at least 97%, at least 97.5%, or    at least 98% (area % as determined by HPLC).

-   C23. The process of any one of embodiments C to C22 wherein compound    Formula IIIa is

-   D. A method for treating cancer in a patient wherein the cancer is    characterized by the overexpression of PI3 kinase, the method    comprising administering a therapeutically effective amount of a PI3    kinase inhibitor compound of Formula III according to embodiment A    to a person in need of such treatment.-   D1. The method of embodiment D wherein the PI3 kinase inhibitor    compound is compound IIIat of the formula:

-   D2. The method of embodiment D or embodiment D1 wherein the dose of    the PI3 kinase inhibitor compound is from about 0.2 mg/kg/day to    about 1.5 mg/kg/day, from about 0.3 mg/kg/day to about 1 mg/kg/day,    or from about 0.4 mg/kg/day to about 0.75 mg/kg/day.-   D3. The method of any one of embodiments D to D2 wherein the    terminal half-life of the PI3 kinase inhibitor compound in a    plurality of cancer cells is from about 10 hours to about 24 hours,    from about 12 hours to about 22 hours, or from about 15 hours to    about 20 hours after a single dose administered on the first day of    a dosage cycle.-   D4. The method of any one of embodiments D to D3 wherein the time to    maximum plasma concentration for the PI3 kinase inhibitor is from    about 1 hours to about 8 hours, from about 2 hours to about 6 hours,    from about 2 hours to about 4 hours, or from about 2 hours to about    3 hours after a single dose administered on the first day of a    dosage cycle.-   D5. The method of any one of embodiments D to D4 wherein the maximum    plasma concentration for the PI3 kinase inhibitor is from about 0.01    μM to about 0.5 μM, from about 0.05 μM to about 0.4 μM, or from    about 0.1 μM to about 0.3 μM after a single dose administered on the    first day of a dosage cycle.-   D6. The method of any one of embodiments D to D5 wherein area under    the concentration-time curve in a plurality of cancer cells from    time 0 to infinity for the PI3 kinase inhibitor is from about 0.2    μM*hr to about 10 μM*hr, from about 0.5 μM*hr to about 10 μM*hr,    from about 1 μM*hr to about 8 μM*hr, or from about 2 μM*hr to about    6 μM*hr after a single dose administered on the first day of a    dosage cycle.-   D7. The method of any one of embodiments D to D6 wherein the area    under the concentration curve in a plurality of cancer cells for the    PI3 kinase inhibitor from time 0 to 24 hours is from about 0.1 μM*hr    to about 10 μM*hr, from about 0.5 μM*hr to about 5 μM*hr, from about    1 μM*hr to about 5 μM*hr, or from about 2 μM*hr to about 4 μM*hr    after a single dose administered on the first day of a dosage cycle.-   D8. The method of any one of embodiments D to D7 wherein the PI3    kinase inhibitor is administered orally.-   D9. The method of any one of embodiments D to D8 wherein the PI3    kinase inhibitor is administered orally without food or under    fasting conditions.-   D10. The method of any one of embodiments D to D9 wherein the cancer    is a brain cancer.-   D11. The method of any one of embodiments D to D10 wherein the    cancer is glioma.-   D12. The method of any one of embodiments D to D10 wherein the    cancer is glioblastoma.-   D13. The method of any one of embodiments D to D12 wherein the    method further comprises administering to the patient an additional    therapeutic agent selected from a chemotherapeutic agent, an    anti-angigenesis therapeutic agent, an anti-inflammatory agent, an    immunomodulatory agent, a neurotropic factor, an agent for treating    cardiovascular disease, an agent for treating liver disease, an    anti-viral agent, an agent for treating blood disorders, an agent    for treating diabetes, and an agent for treating immunodeficiency    disorders.-   D14. The method of D13 wherein the additional therapeutic agent is    bevacizumab.-   D15. The method of D13 wherein the additional therapeutic agent is    temozolomide.

What is claimed is:
 1. A process for preparing a compound of FormulaIIa, from a compound of Formula I in a reaction mixture according to thefollowing reaction scheme:

the process comprising: (i) forming a reaction mixture comprising thecompound of Formula I, organic halide, solvent system, phase transfercatalyst, and base, (ii) reacting the reaction mixture to form areaction product mixture comprising the compound of Formula IIa, and(iii) isolating the compound of Formula IIa from the reaction productmixture, wherein the solvent system comprises at least 5 v/v % water; Xis halide; each R⁵, R¹⁰ and R¹⁵ are independently selected from H orC₁-C₁₀ hydrocarbyl, wherein each hydrocarbyl is optionally substituted,two geminal R⁵, R¹⁰ and/or R¹⁵ groups are oxo, or two geminal R⁵, R¹⁰and/or R¹⁵ groups form a 3, 4, 5, 6, or 7-membered carbocyclyl orheterocyclyl, wherein the carbocyclyl or heterocyclyl is optionallysubstituted; mor is selected from:

wherein mor is optionally substituted with one or more R⁷ groupsindependently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃,—CH(CH₃)₂, —C(CH₃)₃, —CH₂OCH₃, —CHF₂, —CN, —CF₃, —CH₂OH, —CH₂OCH₃,—CH₂CH₂OH, —CH₂C(CH₃)₂OH, —CH(CH₃)OH, —CH(CH₂CH₃)OH, —CH₂CH(OH)CH₃,—C(CH₃)₂OH, —C(CH₃)₂OCH₃, —CH(CH₃)F, —C(CH₃)F₂, —CH(CH₂CH₃)F,—C(CH₂CH₃)₂F, —CO₂H, —CONH₂, —CON(CH₂CH₃)₂, —COCH₃, —CON(CH₃)₂, —NO₂,—NH₂, —NHCH₃, —N(CH₃)₂, —NHCH₂CH₃, —NHCH(CH₃)₂, —NHCH₂CH₂OH,—NHCH₂CH₂OCH₃, —NHCOCH₃, —NHCOCH₂CH₃, —NHCOCH₂OH, —NHS(O)₂CH₃,—N(CH₃)S(O)CH₃, ═O, —OH, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃,—NHC(O)NHCH₂CH₃, —S(O)CH₃, —S(O)CH₂CH₃, —S(O)₂CH₃, —S(O)₂NH₂,—S(O)₂NHCH₃, —S(O)₂N(CH₃)₂, and —CH₂S(O)₂CH₃; and wherein in Formula IR²⁰ is —OH or —NHR²¹, wherein R²¹ is as defined for R⁵and wherein inFormula IIa R²⁰ is —O— or —NR²¹—.
 2. The process of claim 1, wherein thesolvent system comprises at least 50 v/v % water.
 3. The process ofclaim 1, wherein the base is selected from K₃PO₄, Cs₂CO₃, K₂CO₃, KOAc,NaOAc, Na₂CO₃ and KOH.
 4. The process of claim 1, wherein the phasetransfer catalyst is selected from: a quaternary ammonium salt and aphosphonium salt.
 5. The process of claim 4, wherein the phase transfercatalyst is selected from: tetra-n-butylammonium bromide,benzyltrimethylammonium chloride, benzyltriethylammonium chloride,methyltricaprylammonium chloride, methyltributylammonium chloride andmethyltrioctylammonium chloride.
 6. The process of claim 5, wherein thephase transfer catalyst is tetra-n-butylammoniumbromide.
 7. The processof claim 1, wherein the organic halide and the base are present in aboutequimolar amounts.
 8. The process of claim 1, wherein the reactiontemperature is from about 40° C. to about 90° C.
 9. The process of claim1, further comprising admixing a polar protic solvent with the reactionproduct mixture followed by reducing the temperature of the admixture toinduce crystallization of compound Formula IIa in the reaction productmixture, wherein the crystallized compound Formula IIa is isolated fromthe reaction product mixture.
 10. The process of claim 9, wherein thepolar protic solvent is selected from: water, methanol, ethanol,isopropanol, n-propanol, and acetic acid.
 11. The process of claim 10,wherein the polar protic solvent is ethanol.
 12. The process of claim11, wherein the volume ratio of the solvent system to ethanol is fromabout 1:5 v/v to about 5:1 v/v.
 13. The process of claim 11, furthercomprising adding compound Formula IIa seed crystals to the admixture ofthe reaction product mixture and ethanol.
 14. The process of claim 1,wherein the yield of compound Formula IIa is at least 60%.
 15. Theprocess of claim 1, wherein the purity of compound Formula IIa is atleast 97%.
 16. The process of claim 1, wherein each R⁵, R¹⁰ and R¹⁵ isindependently selected from H and optionally substituted C₁₋₅ alkyl, ortwo geminal R⁵, R¹⁰ and/or R¹⁵ groups together are oxo or form a 3 to6-membered cycloalkyl or heterocycloalkyl having one or two hetero atomsselected from N and O.
 17. The process of claim 1, wherein each R⁵, R¹⁰and R¹⁵ is independently selected from H, C₁₋₅ alkyl and C₁₋₅ alkylsubstituted with at least one of deuterium, halogen and hydroxyl. 18.The process of claim 1, wherein in formula I R²⁰ is —OH, —NH₂ or—NH—C₁₋₅ alkyl.
 19. The process of claim 18, wherein in formula I R²⁰ is—OH.
 20. The process of claim 1, wherein the organic halide is1,2-dibromoethane.
 21. The process of claim 20, wherein the molar ratioof the 1,2-dibromoethane to the compound of Formula I is from greaterthan 2:1 to about 4:1.
 22. The process of claim 1, wherein the compoundof Formula IIa is:


23. The process of claim 1, further comprising preparing a compound ofFormula IIIa from a compound of Formula IIa in a reaction mixtureaccording to the following reaction scheme:

wherein preparing a compound of Formula IIIa comprises: (i) forming areaction mixture comprising the compound of Formula IIa,organoboron-R⁴the solvent system comprising at least 5 v/v % water, thebase and the catalyst; (ii) reacting the reaction mixture to form areaction product mixture comprising the compound of Formula IIIa; and(iii) isolating the compound of Formula IIIa from the reaction productmixture, and wherein R⁴ is selected from C₆-C₂₀ aryl, C₂C₂₀ heterocyclyland C₁-C₂₀ heteroaryl, each of which are optionally substituted with oneor more groups independently selected from F, Cl, Br, I, —CH₃, —CH₂CH₃,—CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH₂CH₃, —CH₂CN, —CN, —CF₃, —CH₂OH, —CO₂H,—CONH₂, CONH(CH₃), —CON(CH₃)₂, —NO₂, —NH₂, —NHCH₃, —NHCOCH₃, —OH, —OCH₃,—OCH₂CH₃, —OCH(CH₃)₂, —SH, —NHC(O)NHCH₃, —NHC(O)NHCH₂CH₃, —NHS(O)₂CH₃,—N(CH₃)C(O)OC(CH₃)₃, —S(O)₂CH₃, benzyl, benzyloxy, morpholinyl,morpholinomethyl, and 4-methylpiperazinyl.
 24. The process of claim 1,wherein each R⁵, R¹⁰ and R⁵ are independently selected from H or C₁-C₅hydrocarbyl, wherein each hydrocarbyl is optionally substituted, twogeminal R⁵, R¹⁰ and/or R¹⁵ groups are oxo, or two geminal R⁵, R¹⁰ and/orR¹⁵ groups form a 3, 4, 5, 6, or 7-membered carbocyclyl or heterocyclyl,wherein the carbocyclyl or heterocyclyl is optionally substituted.